U.S. patent application number 10/406031 was filed with the patent office on 2004-03-04 for prothrombin activating protein.
Invention is credited to Jersey, John De, Lavin, Martin, Masci, Paul Pantaleone.
Application Number | 20040043017 10/406031 |
Document ID | / |
Family ID | 28675926 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040043017 |
Kind Code |
A1 |
Masci, Paul Pantaleone ; et
al. |
March 4, 2004 |
Prothrombin activating protein
Abstract
The invention relates to snake venom protease polypeptides and
nucleic acid sequences encoding same. This invention also relates
to methods of making and using the snake venom proteases, e.g., to
promote haemostasis and prevent blood loss such as during surgery
or for treatment of wounds resulting from accidents and other types
of injury or trauma.
Inventors: |
Masci, Paul Pantaleone;
(Brisbane, AU) ; Jersey, John De; (Brisbane,
AU) ; Lavin, Martin; (Brisbane, AU) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
28675926 |
Appl. No.: |
10/406031 |
Filed: |
April 2, 2003 |
Current U.S.
Class: |
424/94.64 ;
435/226 |
Current CPC
Class: |
C12N 9/6408 20130101;
A61L 2400/04 20130101; A61L 15/32 20130101; A61L 17/10 20130101;
A61P 7/04 20180101; A61K 38/00 20130101; A61L 26/0047 20130101;
A61P 43/00 20180101 |
Class at
Publication: |
424/094.64 ;
435/226 |
International
Class: |
A61K 038/48; C12N
009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 3, 2002 |
AU |
PS1483 |
Mar 7, 2003 |
AU |
2003901033 |
Claims
What is claimed:
1. An isolated preparation of a snake venom protease (SVP)
comprising one or more of: a light chain which shares at least 50%
sequence identity with a light chain sequence of any of SEQ ID NOs:
2, 5, 8, 11, 14 or 17, and a heavy chain which shares at least 50%
sequence identity with a heavy chain sequence of any of SEQ ID
NOs:2, 5, 8, 11, 14 or 17, and which does not require calcium for
activity.
2. The isolated preparation of an SVP of claim 1, wherein said SVP
does not require factor Va for activity.
3. The isolated preparation of an SVP of claim 1, wherein said SVP
does not require phospholipid for activity.
4. The isolated preparation of an SVP of claim 1, wherein the SVP
includes a propeptide domain.
5. The isolated preparation of an SVP of claim 1, wherein the SVP
includes an activation domain.
6. The isolated preparation of an SVP of claim 1, wherein the light
and heavy sequences are on the same polypeptide chain.
7. The isolated preparation of an SVP of claim 1, wherein the light
and heavy sequences are on different polypeptide chains.
8. The isolated preparation of an SVP of claim 1, wherein light and
heavy chain proteins are present and are the same or very similar
in length as are naturally occurring species.
9. The isolated preparation of an SVP of claim 1, comprising one or
more of the following domains: a first or propeptide domain which
has at least 31% sequence identity with residues 1-40 of any of the
6 SVP's of FIG. 23, a light chain cleavage site between residues 40
and 41 of any of the 6 SVP's of FIG. 23; a domain which shares at
least 80% sequence identity with residues 41-85 of any of the SVP's
of FIG. 23; a domain which shares at least 75% sequence identity
with residues 86-122 of any of the SVP's of FIG. 23; a domain which
has at least 75% sequence identity with residues 123-165 of any of
the SVP's of FIG. 23; a domain which has at least 75% sequence
identity with residues 166-179 of any of the SVP's of FIG. 23; a
domain which corresponds to residues 180-182 of FIG. 23; a domain
which which has at least 50% sequence identity with residues
183-209 of any SVP of FIG. 23; and a heavy chain domain has at
least 75% sequence identity with residuess 210 -467 (in the case of
the Brown, Coastal Taipan, Inland Taipan, or Red Belly Black
sequence) or 210 456 (in the case of the Tiger and Rough Scale
sequence) of FIG. 23.
10. The isolated preparation of an SVP of claim 1, which comprises
residues H.sub.251, D.sub.309 and S.sub.406 of FIG. 23.
11. The isolated preparation of an SVP of claim 1, which comprises
a sequence which is the same as or differes at no more than 5
residues from the sequence of amino acids 292-305 of any of the
SVP's of FIG. 23.
12. The isolated preparation of an SVP of claim 1, comprising a
dimeric molecule of a fully processed light chain and heavy
chain.
13. The isolated preparation of an SVP of claim 1, comprising a
dimeric molecule of a light and a heavy chain having intrachain
Cys-Cys linkages between 57 and 62, 90and 101, 95 and 110, 112 and
121, 129and 140, and 151 and 164of the light chain, intra chain
Cys-Cys linkages between 216 and 221, 236 and 252, 377 and 391, and
402 and 430 of the heavy chain, and inter chain Cys-Cys linkages
between 172 of the light chain and 329 of the heavy chain.
14. The isolated preparation of an SVP of claim 1, comprising one
or more and in some cases all of the following domains (the
numbering refers to the consensus numbering in FIG. 22): a first or
propeptide domain having at least 30% sequence identity with to
residues 1-40 of any of the SVP's of FIG. 22; a domain having at
least 90% sequence identiy with to residues 41-120 of any of the
SVP's of FIG. 22; a domain having at least 60% sequence identity
with to residues 121-132 of any of the SVP's of FIG. 22; a domain
having at least 80% sequence identity with to residues 1331-182 of
any of the SVP's of FIG. 22; a domain having at least 90% sequence
identity with to residues 183-233 of any of the SVP's of FIG. 22; a
domain having at least 80% sequence identity with to residues
234-378 of any of the SVP's of FIG. 22; a domain having at least
80% sequence identity with to residues 395-456 of any of the SVP's
of FIG. 22; a domain having at least 90% sequence identity with to
residues 457-467 of FIG. 22.
15. The isolated preparation of an SVP of claim 1, wherein said SVP
is a complete activator of prothrombin and having one or more of
the following characteristics: the sequence will be other than S at
residue 41 (all references are to the consensus numbering of FIG.
21), I at residue 48, P at residue 50, N at residue 74, P at
residue 104, N at residue 105, K at residue 123, Q at residue 127,
R at residue 142, SET at residues 145-7, S at residue 154, R at
residue 156, V at residue 159, E at residue 167, D at residue 169,
A at residue 178; will include at least one residue from the
sequence 181-208 any of the Brown, Taipan, Red Belly, Tiger, Rough
Scale sequences of FIG. 21 (or a corresponding residue from Taipan
Inland); will be other than I at residue 228, N at residue 229, G
at residue 232, E at residue 232, H at residue 245, SV at residues
258-9; will include at least one residue from the sequence 260-270
any of the Brown, Taipan, Red Belly, Tiger, Rough Scale sequences
of FIG. 21 (or a corresponding residue from Taipan Inland); will be
other than R at residue 274, T at residue 286, NYYY-VHQN at
residues 292-300, R at residue 303, A at residue 305, R at residues
314, E at residue 339, S at residue 345, RIQFKQPT at residues
353-360, I at residue 367, T at residue 368, D at residues 382, R
at residue 384, Q at residue 387, N at residues 389, I at residue
424, R at residue 342, K at residues 451, SL at residue 454-455; or
will include at least one residue from the sequence 457-467 of any
of the Brown, Taipan, Red Belly, Tiger, Rough Scale sequences of
FIG. 21 (or a corresponding residue from Taipan Inland);
16. The isolated preparation of an SVP of claim 1, wherein said SVP
is a partially complete activator of prothrombin and having one or
more of the following characteristics: the sequence will include at
least one residue from the sequence 181-208 any of the Brown,
Taipan, Red Belly, Tiger, Rough Scale sequences of FIG. 21 (or a
corresponding residue from Taipan Inland); or will include at least
one residue from the sequence 260-270 any of the Brown, Taipan, Red
Belly, Tiger, Rough Scale sequences of FIG. 21 (or a corresponding
residue from Taipan Inland).
17. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a light chain having at least 95% sequence
identity with a light chain sequence from any of SEQ ID NOs:2, 5,
8, 11, 14 or 17.
18. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a light chain which differs at 10 or fewer
residues form a light chain sequence from any of SEQ ID NOs:2, 5,
8, 11, 14 or 17.
19. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a light chain having a sequence from any of
SEQ ID NOs:2, 5, 8, 11, 14 or 17.
20. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a heavy chain having at least 95% sequence
identity with a heavy chain sequence from any of SEQ ID NOs:2, 5,
8, 11, 14 or 17.
21. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a heavy chain which differs at 10 or fewer
residues form a heavy chain sequence from any of SEQ ID NOs:2, 5,
8, 11, 14 or 17.
22. The isolated preparation of an SVP of claim 1, wherein the
preparation comprises a heavy chain having a sequence from any of
SEQ ID NOs:2, 5, 8, 11, 14 or 17.
23. An isolated nucleic acid selected from the group consisting of:
a) a nucleic acid sequence which encodes a polypeptide comprising
the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14 or 17; b) a
nucleic acid molecule comprising the nucleotide sequence shown in
SEQ ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18, or a full
complement of SEQ ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or
18; c) a nucleic acid molecule having at least 85% sequence
identity with the nucleotide sequence shown in SEQ ID NOs:1, 3, 4,
6, 7, 9, 10, 12, 13, 15, 16 or 18. d) a nucleic acid molecule
encoding a polypeptide having amino acid residues 41 to 179 of any
of the six SVPs of FIG. 23; e) a nucleic acid molecule encoding a
polypeptide having amino acid residues 210 to 467, in the case of a
Brown, Costal Taipan, Inland Taipan or Red Belly sequence of FIG.
29 or residues 210 to 456, in the case of a Tiger or Rough Scale
sequence of FIG. 23; f) a nucleic acid molecule encoding a
polypeptide having amino acid residues 1 to 40 of any of the 6 SVPs
of FIG. 23; g) a nucleic acid molecule encoding a polypeptide
having amino acid residues 180 to 209 or residues 183 to 209 of any
of the 6 SVPs of FIG. 23.
24. The nucleic acid molecule of claim 23, further comprising
vector nucleic acid sequences.
25. The nucleic acid molecule of claim 23, further comprising
nucleic acid sequences encoding a heterologous polypeptide.
26. A vector comprising the nucleic acid molecule of claim 23.
27. A host cell which contains the nucleic acid molecule of claim
23.
28. A method for producing an SVP polypeptide, the method
comprising culturing the host cell of claim 27 under conditions in
which the nucleic acid molecule is expressed.
29. A composition comprising a polypeptide of claim 1, wherein the
pH of the composition is between about 5 and 9.
30. The composition of claim 29, wherein the pH of the composition
is about 6.5 to 7.
31. A composition comprising a polypeptide of claim 1 and a
polyol.
32. The composition of claim 31, wherein the polyol is
glycerol.
33. A pharmaceutical composition comprising a polypeptide of claim
1 and a pharmaceutically acceptable carrier.
34. A kit comprising a polypeptide of claim 1, and one or more of:
instructions for use; other reagents; a diluent; devices or other
materials for preparing the snake venom protease for
administration; pharmaceutically acceptable carriers; and devices
or other materials for administration to a subject.
35. The kit of claim 34, wherein the kit comprises one or more
reagents selected from the group consisting of: a cofactor, an
anti-microbial, e.g., an antibiotic, an antiviral, an antifungal,
an antiparasitic agent, an anti-inflammatory agent, an
antihistamine, an anti-fibrolytic agent, an analgesic ,and a growth
factor.
36. The kit of claim 35, further comprising one or more cofactors
selected from the group consisting of: calcium, a phospholipid, and
factor Va.
37. A method of treating a subject comprising administering an SVP
of claim 1 to said subject, thereby treating said subject.
38. The method of claim 37, wherein the method inhibits bleeding
from a site on or in the subject's body.
38. The method of claim 38, wherein said site is the site of a
medical or surgical intervention.
39. The method of claim 38, wherein said site is the site of
unwanted trauma.
40. The method of claim 38, wherein said subject has a deficiency
in the ability to form or maintain a blood clot.
41. The method of claim 40, wherein said deficiency is due to a
genetic defect or the result of the administration of a drug which
reduced the ability of the subject to form or maintain a blood
clot.
42. The method of claim 37, wherein said SVP is administered by a
person other than the subject.
43. The method of claim 37, wherein said SVP is self
administered.
44. The method of claim 37, wherein said SVP is provided to the
subject in advance of a need to use it.
45. The method of claim 37, wherein said SVP is provided in a
liquid resistant container along with instructions for its use.
46. A preparation of the SVP of claim 1 disposed in a liquid or gas
impermeable container.
47. The preparation of claim 46, wherein said container is formed
so as to allow dispensing of SVP in liquid, spray, aerosol,
powdered, or crystalline form.
48. The preparation of claim 47, wherein said container is formed
so as to allow dispensing of a metered or predetermined dosage of
SVP.
49. A device upon which is disposed an amount of SVP of claim 1
sufficient to inhibit bleeding when the device is brought in
contact with a subject.
50. The device of claim 49, wherein said device is any of a
bandage, compress, wound dressing, suture, or an article of
clothing.
51. Machine-readable medium on which is recorded the nucleic acid
or protein sequence of an SVP of claim 1 or 23.
52. A method of analyzing an SVP sequence comprising providing an
SVP nucleic acid or amino acid sequence and comparing the SVP
sequence with a second sequence or transmitting said sequence from
one computer to a second computer, to thereby analyze SVP.
53. A nucleic acid library from any of a brown, inland Taipan,
coastal Taipan, red belly, tiger, or rough scale snake.
54. A protein library from any of a brown, inland Taipan, coastal
Taipan, red belly, tiger, or rough scale snake.
55. An isolated polypeptide comprising the sequence:
MAPQLLLCLILTFLWSLPEAESNVFLKSKX.sub.1ANRFLQRTKRX.sub.2NSLX.sub.3EEX.sub.4X-
.sub.5X.sub.6G
NIERECIEEX.sub.7CSKEEAREX.sub.8FX.sub.9DX.sub.10EKTEX.sub.1-
1IFWNVYVDGDQCSSNPCHYX.sub.12GX.sub.13CKDGIGSYTCTCLX.sub.14X.sub.15YEGKNCEX-
.sub.16X.sub.17LX.sub.18X.sub.19SCRX.sub.20X.sub.21NGNCWHFCKX.sub.22V
QX.sub.23X.sub.24X.sub.25QCSCAEX.sub.26YX.sub.27LGX.sub.28DGHSCVAX.sub.29-
GX.sub.30FSCGRNIKX.sub.31RNKREASLP
DFVQSX.sub.32X.sub.33AX.sub.34X.sub.35K-
KSDNPSPDIRIX.sub.36NGMDCKLGECPWQAX.sub.37LX.sub.38X.sub.39X.sub.40X.sub.41-
X.sub.42X.sub.43X.sub.44FCGGTILSPIX.sub.45VLTAAHCIX.sub.46X.sub.47X.sub.48-
X.sub.49X.sub.50X.sub.51SVX.sub.52VGEIX.sub.53X.sub.54SRX.sub.55X
.sub.56X.sub.57X.sub.58X.sub.59LLSVDK
X.sub.60YVHX.sub.61KFVX.sub.62X.sub-
.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70X.sub.71X.sub.7-
2X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77YDYDIAIX.sub.78X.sub.79
X.sub.80KTPIQFSENVVPACLPTADFAX.sub.81X.sub.82VLMKQDX.sub.83GIX.sub.84SGFG-
X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89X.sub.90
X.sub.91X.sub.92SX.sub.93-
X.sub.94LKX.sub.95X.sub.96X.sub.97VPYVDRHTCMX.sub.98SSX.sub.99X.sub.100X.s-
ub.101ITX.sub.102X.sub.103MFCAGYDT LP
X.sub.104DACQGDSGGPHITAYXI.sub.105DT-
HFX.sub.106TGIX.sub.107SWGEGCAX.sub.108X.sub.109GX.sub.110YGX.sub.111Y
TKX.sub.112SX.sub.113FIX.sub.114WIKX.sub.115X.sub.116MX.sub.117X.sub.118X-
.sub.119Z wherein X.sub.1, X.sub.10, X.sub.12-13, X.sub.15-16,
X.sub.19-23, X.sub.25, X.sub.27-30, X.sub.33-34, X.sub.37,
X.sub.39, X.sub.42-47, X.sub.50, X.sub.53-56, X.sub.58-62,
X.sub.64, X.sub.79, X.sub.81-83, X.sub.85-94, X.sub.96,
X.sub.99-105, X.sub.108-109, X.sub.113-115 and X.sub.117-119 are
each independently selected from any amino acid residue; each of
X.sub.2, X.sub.6, X.sub.11, X.sub.14, X.sub.26, X.sub.31,
X.sub.48X.sub.57 and X.sub.63 is a small amino acid residue; each
of X.sub.3, X.sub.4, X.sub.8, X.sub.17, X.sub.18, X.sub.35-36,
X.sub.38, X.sub.51-52, X.sub.78, X.sub.80, X.sub.84, X.sub.95,
X.sub.98, X.sub.106-107, X.sub.111-112 and X.sub.116 is a
hydrophobic amino acid residue; each of X.sub.5, X.sub.7 and
X.sub.110 is a basic amino acid residue; each of X.sub.9,
X.sub.40-41 and X.sub.49 is a charged amino acid residue; X.sub.24
is an acidic amino acid residue; X.sub.32 is a neutral/polar amino
acid residue; X.sub.65-67, X.sub.70-72 and X.sub.75 are each
independently absent or selected from any amino acid residue;
X.sub.68 and X.sub.74 are each independently absent or selected
from acidic amino acid residues; X.sub.69, X.sub.73 and X.sub.76
are each independently absent or selected from hydrophobic amino
acid residues; X.sub.77 is absent or is a small amino acid residue;
and Z is absent or is a peptide of from 1-20 amino acids
Description
[0001] This application claims the benefit of a previously filed
Australian Provisional Application Nos. PS1483, filed Apr. 3, 2002,
and 2003901033, filed Mar. 7, 2003, the contents of which are
incorporated in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to novel snake venom protease
polypeptides and nucleic acid sequences encoding same. This
invention also relates to methods of making and using the snake
venom proteases, e.g., to promote haemostasis and prevent blood
loss such as during surgery or for treatment of wounds resulting
from accidents and other types of injury or trauma.
BACKGROUND OF THE INVENTION
[0003] Haemostasis, commonly referred to as blood coagulation or
blood clotting, is a key biological response to wounding or injury
that prevents excessive blood loss. The biochemical cascade that
controls haemostasis in mammals is well understood. A crucial step
in this pathway is the activation of prothrombin by a
prothrombinase complex to produce thrombin, which in turn activates
Factor XIIIa, which cross-links fibrin to form a stable clot
(Stubbs & Bode, 1994, Curr. Opin. Struct. Biol. 4 823-32).
[0004] In mammals, the prothrombin activator complex in vivo
typically consists of a serine proteinase factor Xa and a cofactor
Va formed on phospholipid membranes in the presence of calcium ions
(Suttie & Jackson, 1977, Physiol. Rev. 57 1). The mammalian
prothrombinase complex consists of a cofactor, Factor Va, and a
serine protease, Factor Xa. Factor Xa alone activates prothrombin
very slowly, however, in the presence of accessory proteins
including the nonenzymatic cofactor Factor Va, calcium ions
(Ca.sup.2+) and phospholipid, prothrombin activation is enhanced
many fold. In vivo, Factor Xa binds the phospholipid membrane of
blood platelets by gamma-carboxyglutamic acid residues and has
preferential cleavage for Arg274-Thr275 followed by Arg323-Ile324
bonds in prothrombin to form thrombin.
[0005] Given the importance of controlling blood loss during
surgery or following injury or trauma, the identification of
regulators that either promote blood clotting or inhibit the
dissolution of clots (such as by the fibrinolytic
plasmin/plasminogen pathway; Royston et al., 1990, Blood Coagul.
Fibrinol. 1 53; Orchard et al., 1993, Br. J. Haematol. 85 596) has
become an area of intense interest.
[0006] In particular, snake venoms have become useful sources of
proteins that can either prevent fibrinolysis or promote blood
clotting, as a result of blood loss during surgery, trauma in
mammals.
[0007] For example, inhibitors of fibrinolysis have been isolated
from venom of the Australian common brown snake Pseudonaja textilis
(International Publication WO 99/58569). With regard to snake
venom-derived prothrombin activators, reference is also made to
Chinese Patent 1298017 which discloses prothrombin activators
isolated from venom of the Taipan snake Oxyuranus scutellatus:
prothrombin activating enzyme (designated Os-II) and activated
factor Xa. The Chinese group proposed that to promote haemostasis
such as in the case of a bleeding wound, Os-II is optimally added
one hour before addition of factor Xa to thereby activate
prothrombin. They proposed that the simultaneous action of the two
can activate prothrombin and raise the yield of thrombin.
[0008] Reference is also made to Joseph et al., 1999, Blood 94 621
which discloses a factor Xa-like prothrombin activator (trocarin)
isolated from the venom of the Australian rough-scaled snake
Tropidechis carinatus. Trocarin forms a prothrombin activator
complex that catalyzes formation of thrombin from prothrombin in
vitro in the presence of phospholipid, factor Va and calcium
ions.
[0009] Current haemostatic agents use bovine or human derived blood
product components to replace various factors to prevent
fibrinolysis or promote blood clotting, as a result of blood loss
during surgery, trauma in mammals. The use of bovine or human
derived blood product components may potentially expose patients to
viral contamination or other adverse events.
SUMMARY OF THE INVENTION
[0010] The invention is based, in part, on the discovery of
prothrombin activating polypeptides, referred to herein as "snake
venom proteases or SVP's," which are factor independent. The snake
venom proteases share certain amino acid sequences similarity to
the amino acid sequences of factor Xa and trocarin which are
prothrombin activators that require calcium, phospholipids and
factor Va for activation. However, the snake venom proteases of the
invention are complete or partially complete prothrombin activators
and thus do not have the cofactor requirements of human factor Xa
or trocarin. In other words, they can process prothrombin to
thrombin in the absence of cofactors such as calcium, phospholipids
and/or factor Va. For example, snake venom proteases from brown,
coastal taipan and inland taipan venom are complete prothrombin
factors in that they can process prothrombin to thrombin in the
absence of calcium, phospholipids and factor Va. These SVP's appear
to include an internal domain, residues 292-305 of FIG. 23, which
makes them independent of host supplied Factor Va. Snake venom
proteases from, for example, red belly, tiger and rough scale snake
venom are partially complete prothrombin activators in that they
can process prothrombin in the absence of calcium and phospholipids
but require the presence of factor Va. In addition, preferred SVP's
of the invention can cleave descarboxy prothrombin, which is a poor
substrate for human factor X.
[0011] Accordingly, in one aspect, the invention features snake
venom protease polypeptides, and biologically active or antigenic
fragments thereof, that are complete or partially complete
prothrombin activators and that are useful, e.g., as reagents to
increase coagulation. In another embodiment, the invention provides
snake venom protease polypeptides having prothrombin activating
activity.
[0012] In one embodiment, the snake venom protease includes one or
more of a light chain and a heavy chain or biologically active
fragments thereof. Preferred light and heavy chain proteins are the
same or very similar (differing, e.g., by 1 or 2 residues) in
length as naturally occurring species. In another embodiment, the
snake venom proteases include a propeptide, a light chain, an
activation peptide and a heavy chain. All processing intermediates,
whether or not present in nature, are within the invention. Thus,
in yet another embodiment, the snake venom protease polypeptides of
the invention include a light chain, an activation peptide and a
heavy chain. The preferred embodiment includes a light chain and
heavy chain from which the propeptide domain and activation peptide
or peptides have been cleaved. Purified preparations can include or
have the cleaved propeptide domains and cleavage fragments purified
away.
[0013] In a preferred embodiment, the complete or partially
complete prothrombin activating SVP includes one or more and in
some cases all of the following domains (the numbering refers to
the consensus numbering in FIG. 23):
[0014] a first or propeptide domain which corresponds to residues
1-40 of FIG. 23. In preferred embodiments, this domain can have at
least 31, 40, 80, 90, 95, or 98% sequence similarity with, or
differ at no more than 1, 2, 3, 5, or 10 amino acid residues from,
the corresponding domain of any of the 5sequences presented in FIG.
29, and in particular to the corresponding domain of one of the
complete SVP's, namely the Brown, Coastal Taipan, or Inland Taipan
sequence, or one of the partially complete SVP's, namely the Red
Belly Black, Tiger, or Rough Scale. Preferred active products will
of course lack the propeptide domain. It may in some cases be
desirable to modify the snake propeptide domain to make it more
similar to the propeptide domain of human factor X, or to replace
the snake propeptide domain with a human propeptide domain. The
propeptide domains are 100% conserved in all 6 snakes with the
exception of a single amino acid change V.fwdarw.E in the Red
Bellied Black. Comparison with the corresponding human sequence
reveals 12/40 identical residues (30% identity). The majority of
the conserved residues are hydrophobic;
[0015] a light chain cleavage site between residues 40 and 41 of
FIG. 23;
[0016] a domain which corresponds to residues 41-85 of FIG. 23.
This domain may be functionally analogous to the GLA (gamma carboxy
glutamic acid) domain of human factor X. In preferred embodiments,
this domain can have at least 71, 75, 80, 85, 90, 95 or 98%
sequence similarity with, or differ at no more than 1, 2, 3, 5, or
10 amino acid residues from, the corresponding domain of any of the
6 sequences presented in FIG. 23, and in particular to the
corresponding domain of one of the complete SVP's of, namely the
Brown, Coastal Taipan, or Inland Taipan sequence, or one of the
partially complete SVP's, namely the Red Belly Black, Tiger, or
Rough Scale. In some embodiments, it may be desirable to conserve
one or more of the 11 glutamic acid residues in this region. Ten of
these are conserved between the human factor X sequence and all 6
of the snake sequences including residues 46/47, 54, 56, 59/60
65/66, 69, 72. Note that 79 is also gamma-carboxylated in human and
there are 2 other potential sites in all 6 snake sequences of FIG.
23 at residues 76 and 78. In many embodiments, the initial residue
of this domain is the initial residue of the light chain of the
product. In a preferred embodiment, this domain shares at least 85%
sequence identity with the corresponding domain of one of the six
snake venom proteases disclosed herein;
[0017] a domain which corresponds to residues 86-122 of FIG. 23.
This domain may be functionally analogous to the first EGF domain
of human factor X. In preferred embodiments, this domain can have
at least 71, 75, 80, 90, 95 or 98% sequence similarity with, or
differ at no more than 1, 2, 3, 5, or 10 amino acid residues from,
the corresponding domain of any of the 6 sequences presented in
FIG. 23, and in particular to the corresponding domain of one of
the complete SVP's of, namely the Brown, Coastal Taipan, or Inland
Taipan sequence, or one of the partially complete SVP's, namely the
Red Belly Black, Tiger, or Rough Scale. Identity with snake
consensus is 25/37. The domain has 70% identity with the human
sequence. In a preferred embodiment, this domain shares at least
70% sequence identity with the corresponding domain of one of the
six snake venom proteases disclosed herein;
[0018] a domain which corresponds to residues 123-165 from any of
the 6 snake sequences of FIG. 23. This domain may be functionally
analogous to the second EGF domain of human factor X. In preferred
embodiments, this domain can have at least 36, 50, 75, 80, 90, 95
or 98% sequence similarity with, or differ at no more than 1, 2, 3,
5, or 10 amino acid residues from, the corresponding domain of any
of the 6 sequences presented in FIG. 23, and in particular to the
corresponding domain of one of the complete SVP's of, namely the
Brown, Coastal Taipan, or Inland Taipan sequence, or one of the
partially complete SVP's, namely the Red Belly Black, Tiger, or
Rough Scale. Identity with snake consensus is 15/43. The domain as
35% identity with the human sequence. In a preferred embodiment,
this domain shares at least 50% sequence identity with the
corresponding domain of one of the six snake venom proteases
disclosed herein;
[0019] a domain which corresponds to residues 166-179 from among
the 6 snake sequences of FIG. 23. In preferred embodiments, this
domain can have at least 75, 80, 90, 95 or 98% sequence similarity
with, or differ at no more than 1, 2, 3, 5, or 10 amino acid
residues from, the corresponding domain of any of the 6 sequences
presented in FIG. 23, and in particular to the corresponding domain
of one of the complete SVP's of, namely the Brown, Coastal Taipan,
or Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 70% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0020] a domain which corresponds to residues 180-182 of FIG. 23.
In preferred embodiments, this domain can have at least 1, 2, or 3
resides which are the same as seen in any of the 6 sequences
presented in FIG. 23. This domain is preferably absent in an active
product;
[0021] a domain which corresponds to residues 183-209 of FIG. 23.
This domain may be functionally analogous to the activation peptide
in human factor X. In preferred embodiments, this domain can have
at least 17, 50, 75, 80, 90, 95 or 98% sequence similarity with, or
differ at no more than 1, 2, 3, 5, or 10 amino acid residues from,
the corresponding domain of any of the 6 sequences presented in
FIG. 23, and in particular to the corresponding domain of one of
the complete SVP's of, namely the Brown, Coastal Taipan, or Inland
Taipan sequence, or one of the partially complete SVP's, namely the
Red Belly Black, Tiger, or Rough Scale. Identity with snake
consensus sequences is 8/51. There is 16% identity with the human
sequence. This is the region that is cleaved out when processing
the light and heavy chains of the protease, and is preferably not
present in active products. The sequence is 51 amino acids for
human factor X and 27 for each of the snakes. In a preferred
embodiment, this domain shares at least 50% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0022] a heavy chain which corresponds to residues 210-467 (in the
case of the Brown, Coastal Taipan, Inland Taipan, or Red Belly
Black sequence) or 456 (in the case of the Tiger and Rough Scale
sequence) of FIG. 23. This domain may be functionally analogous to
the heavy chain in human factor X. In preferred embodiments, this
domain can have at least 50, 75, 80, 90, 95 or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 6
sequences presented in FIG. 23, and in particular to the
corresponding domain of one of the complete SVP's of, namely the
Brown, Coastal Taipan, or Inland Taipan sequence, or one of the
partially complete SVP's, namely the Red Belly Black, Tiger, or
Rough Scale. Identity with snake consensus sequences is 135/268
giving a 50% identity with the human sequence. The catalytic domain
of human factor X contains an essential active site triad
H.sub.236, D.sub.282 and S.sub.379. These 3 residues are conserved
in all 6 snakes as H.sub.251, D.sub.309 and S.sub.406 in FIG. 23
and are conserved in preferred embodiments of the SVP's of the
invention. Amino acids 292-305 appear to contribute factor Va like
activity and the sequence, or one having differing by no more than
1, 2, 3, 4, or 5 residues from a sequence of 292-305 should be
present in complete SVP's. In a preferred embodiment, this domain
shares at least 75% sequence identity with the corresponding domain
of one of the six snake venom proteases disclosed herein.
[0023] As is alluded to above, a preferred embodiment will include
a dimeric molecule of a fully processed light chain and heavy
chain, which have been cleaved from the propeptide domain and
activation or cleavage domains. In preferred embodiments the light
chain includes intra chain Cys-Cys linkages between 57 and 62,
90and 101, 95 and 110, 112 and 121, 129and 140, and/or 151 and
164of the light chain, intra chain Cys-Cys linkages between 216 and
221, 236 and 252, 377 and 391, and/or 402 and 430 of the heavy
chain, and inter chain Cys-Cys linkages between 172 of the light
chain and 329 of the heavy chain. In preferred embodiments, the SVP
is a complete or partially complete prothrombin activator in that
it shows significantly greater activity in the absence of cofactors
than does an incomplete activator, e.g., human factor X or
trocarin. Preferably, the activity of the complete or partially
complete prothrombin activator is at least 1.5, 2, 4, 10, 15, 20,
50, or 100 fold (two orders of magnitude) higher than that of an
incomplete activator, e.g., human factor Xa, or trocarin, alone.
This comparison is made between a snake venom protease and an
incomplete activator measured under the same or similar conditions,
e.g., in the absence of Ca and phospholipids. In preferred
embodiments, the % of activity (i.e., the activity of the complete
or partially complete activator in the absence of Ca and
phospholipid as a % of that seen with the same activator in the
presence of Ca and phospholipids) of a complete or partially
complete is at least 1.5, 2, 4, 10, 15, 20, 50, 100, 1000 or 4000
fold greater than the same % shown by an incomplete activator,
e.g., human factor X or trocarin. Preferred complete or partially
complete activators will clot citrated plasma at concentration of
about 10.sup.-10 to 10.sup.-06 M, e.g., at 10.sup.-8 or 10.sup.-7
M, giving clotting times of about 50 to 15 seconds, demonstrating
Ca.sup.2+ and phospholipid independence. Accordingly, the
prothrombin activator shows kinetic properties of cofactor
independence (calcium ions and/or phospholipid) in the
concentration range of about 10.sup.-10 to 10.sup.-06 M
concentration range being a suitable working range to reduce blood
loss.
[0024] In a preferred embodiment, the complete or partially
complete prothrombin activating SVP includes one or more and in
some cases all of the following domains (the numbering refers to
the consensus numbering in FIG. 22):
[0025] a first or propeptide domain which corresponds to residues
1-40 from among the five snake sequences of FIG. 22 (or the
corresponding sequence of Inland Taipan). In preferred embodiments
this domain can have at least 31, 40, 80, 90, 95, or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 5
sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. Preferred active
products will of course lack the propeptide domain;
[0026] a domain which corresponds to residues 41-120 from the five
snake sequences of FIG. 22 (or the corresponding sequence of Inland
Taipan) having at least 67, 90, 95, or 98% sequence similarity
with, or differs at no more than 1, 2, 3, 5, or 10 amino acid
residues from, the corresponding domain of any of the 5 sequences
presented in FIG. 22 (or the corresponding sequence of Inland
Taipan), and in particular to the corresponding domain of one of
the complete SVP's of, namely the Brown, Coastal Taipan, or Inland
Taipan sequence, or one of the partially complete SVP's, namely the
Red Belly Black, Tiger, or Rough Scale. In a preferred embodiment,
this domain shares at least 90% sequence identity with the
corresponding domain of one of the six snake venom proteases
disclosed herein;
[0027] a domain which corresponds to residues 121-132 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) having at least 43, 60, 65 80, 85, 90, 96, or 98%
sequence similarity with, or differs at no more than 1, 2, 3, 5, or
10 amino acid residues from, the corresponding domain of any of the
5 sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 60% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0028] a domain which corresponds to residues 133-182 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) having at least 80, 85, 90, 96, or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 5
sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 80% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0029] a domain which corresponds to residues 183-233 from among
the snake sequence of FIG. 22 (or the corresponding sequence of
Inland Taipan) having at least 17, 30, 50, 95, 96, or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 5
sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale; Preferred active
products will of course lack the activation domains. In a preferred
embodiment, this domain shares at least 90% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0030] a domain which corresponds to residues 234-378 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) having at least 80, 85, 90, 96, or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 5
sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 80% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0031] a domain which corresponds to residues 379-394 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) having at least 39, 30, 50, 80, 85, 90, 96, or
98% sequence similarity with, or differ at no more than 1, 2, 3, 5,
or 10 amino acid residues from, the corresponding domain of any of
the 5 sequences presented in FIG. 22 (or the corresponding sequence
of Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 50% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0032] a domain which corresponds to residues 395-456 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) having at least 80, 85, 90, 96, or 98% sequence
similarity with, or differs at no more than 1, 2, 3, 5, or 10 amino
acid residues from, the corresponding domain of any of the 5
sequences presented in FIG. 22 (or the corresponding sequence of
Inland Taipan), and in particular to the corresponding domain of
one of the complete SVP's of, namely the Brown, Coastal Taipan, or
Inland Taipan sequence, or one of the partially complete SVP's,
namely the Red Belly Black, Tiger, or Rough Scale. In a preferred
embodiment, this domain shares at least 80% sequence identity with
the corresponding domain of one of the six snake venom proteases
disclosed herein;
[0033] a domain which corresponds to residues 457-467 from among
the five snake sequences of FIG. 22 (or the corresponding sequence
of Inland Taipan) which can be absent, or if present, has at least
90, 96, or 98% sequence similarity with, or differs at no more than
1, 2, 3, or 5 amino acid residues from, the corresponding domain of
any of the 5 sequences presented in FIG. 22 (or the corresponding
sequence of Inland Taipan), and in particular to the corresponding
domain of one of the complete SVP's of, namely the Brown, Coastal
Taipan, or Inland Taipan sequence, or one of the partially complete
SVP's, namely the Red Belly Black, Tiger, or Rough Scale. In a
preferred embodiment, this domain shares at least 90% sequence
identity with the corresponding domain of one of the six snake
venom proteases disclosed herein;
[0034] As is alluded to above, a preferred embodiment will include
a dimeric molecule of a fully processed light chain and heavy
chain, which have been cleaved from the propeptide domain and
activation or cleavage domains. In preferred embodiments the light
chain includes intra chain Cys-Cys linkages between 57 and 62,
90and 101, 95 and 110, 112 and 121, 129and 140, and/or 151 and
164of the light chain, intra chain Cys-Cys linkages between 216 and
221, 236 and 252, 377 and 391, and/or 402 and 430 of the heavy
chain, and inter chain Cys-Cys linkages between 172 of the light
chain and 329 of the heavy chain. In preferred embodiments, the
dimeric SVP is a complete prothrombin activator. In others, it is a
partially complete prothrombin activator. In preferred embodiments,
the SVP is a complete or partially complete prothrombin activator
in that it shows significantly greater activity in the absence of
cofactors than does an incomplete activator, e.g., human factor X
or trocarin. Preferably, the activity of the complete or partially
complete prothrombin activator is at least 1.5, 2, 4, 10, 15, 20,
50, 100, 1000, or 4000 fold (two to four orders of magnitude)
higher than that of an incomplete activator, e.g., human factor Xa,
or trocarin, alone. This comparison is made between a snake venom
protease and an incomplete activator measured under the same or
similar conditions, e.g., in the absence of Ca and phospholipids.
In preferred embodiments, the % of activity (i.e., the activity of
the complete or partially complete activator in the absence of Ca
and phospholipid as a % of that seen with the same activator in the
presence of Ca and phospholipids) of a complete or partially
complete is at least 1.5, 2, 4, 10, 15, 20, 50, 100, 1000, or 4000
fold greater than the same % shown by an incomplete activator,
e.g., human factor X or trocarin. Preferred complete or partially
complete activators will clot citrated plasma at concentration of
about 10.sup.-10 to 10.sup.-06 M, e.g., at 10.sup.-8 or 10.sup.-7
M, giving clotting times of about 50 to 15 seconds, demonstrating
Ca.sup.2+ and phospholipid independence. Accordingly, the
prothrombin activator shows kinetic properties of cofactor
independence (calcium ions and/or phospholipid) in the
concentration range of about 10.sup.-10 to 10.sup.-06 M
concentration range being a suitable working range to reduce blood
loss.
[0035] The SVP's of the invention do not include trocarin, shown
for example in FIG. 21. In preferred embodiments, the processed
light chain of a complete SVP will differ from the processed light
chain of trocarin by at least 1, 3, 5, 10, 15 or 20 residues. In
preferred embodiments, the processed heavy chain of a complete SVP
will differ from the processed heavy chain of trocarin by at least
5, 10, 15, 20 or 30 residues. (differ means differ in identity or
by insertion or deletion, unless otherwise indicated).
[0036] In preferred embodiments, the sequence of a complete SVP of
the invention will have one or more of the following properties, it
will be other than serine at residue 41 (all references are to the
consensus numbering of FIG. 21), isoleucine at residue 48, proline
at residue 50, asparginine at residue 74, proline at residue 104,
asparginine at residue 105, lysine at residue 123, glutamine at
residue 127, arginine at residue 142, serine, glutamic acid,
threonine at residues 145-7, serine at residue 154, arginine at
residue 156, valine at residue 159, glutamic acid at residue 167,
aspartic acid at residue 169, alanine at residue 178; will include
at least one residue from the sequence 181-208 any of the Brown,
Taipan, Red Belly, Tiger, Rough Scale sequences of FIG. 21 (or a
corresponding residue from Taipan Inland); will be other than
isoleucine at residue 228, asparginine at residue 229, glycine at
residue 233, glutamic acid at residue 232, histidine at residue
245, serine, valine at residues 258-9; will include at least one
residue from the sequence 260-270 any of the Brown, Taipan, Red
Belly, Tiger, Rough Scale sequences of FIG. 21 (or a corresponding
residue from Taipan Inland); will be other than arginine at residue
274, threonine at residue 286,
asparganine-tyrosine-tyrosine-tyrosine-valine-histidine-glutamine-asparga-
nine at residues 292-300, arginine at residue 303, alanine at
residue 305, arginine at residue 314, glutamic acid at residue 338,
serine at residue 345, RIQFKQPT at residues 353-360, isoleucine at
residue 367, threonine at residue 368, aspartic acid at residues
382, arginine at residue 384, glutamine at residue 387, asparginine
at residues 389, isoleucine at residue 424, arginine at residue
342, lysine at residues 451, serine, leucine at residue 454-455; or
will include at least one residue from the sequence 457-467 of any
of the Brown, Taipan, Red Belly, Tiger, Rough Scale sequences of
FIG. 21 (or a corresponding residue from Taipan Inland);
[0037] In preferred embodiments, the processed light chain of a
partially complete SVP will differ from the processed light chain
of trocarin by at least 1, 3, 5, 10, or 15 residues. In preferred
embodiments, the processed heavy chain of a complete SVP will
differ from the processed heavy chain of trocarin by at least 5,
10, 15, 20 or 30 residues. In preferred embodiments, the sequence
of a partially complete SVP of the invention will include at least
one residue from the sequence 181-208 any of the Brown, Taipan, Red
Belly, Tiger, Rough Scale sequences of FIG. 21 (or a corresponding
residue from Taipan Inland); or will include at least one residue
from the sequence 260-270 any of the Brown, Taipan, Red Belly,
Tiger, Rough Scale sequences of FIG. 21 (or a corresponding residue
from Taipan Inland).
[0038] In a preferred embodiment, the SVP is a complete prothrombin
activator and includes one or both of a light chain having at least
87, 89 or 90% sequence identity with, or differs at not more than
16, 14, or 13 residues from: the consensus sequence of FIG. 24 or a
heavy chain that has at least 82, 85 and 84% identity or differs at
not more than 45, 39, or 40 residues from the consensus sequence of
FIG. 24.
[0039] In preferred embodiments, the complete SVP includes one or
both light and heavy chain which is identical with or has at least
84, 86 or 86% sequence identity with, or differs at no more than 61
or 53 residues from, the sequence of Brown, Coastal Taipan, or
Inland Taipan SVP sequence shown in FIG. 24.
[0040] In a preferred embodiment the SVP is a partially complete
prothrombin activator and includes one or both of a light and heavy
chain having at least 84% sequence identity with, or differs at not
more than 61 or 53 residues from: the sequence of FIG. 24
[0041] In preferred embodiments the partially complete SVP includes
one or both of a light and heavy chain which is identical with or
has at least 84, 80 or 82% sequence identity with, or differs at no
more than 61, 76, 68 residues from, the sequence of Red Belly
Black, Tiger, or Rough Scale SVP sequence shown in FIG. 24.
[0042] In other embodiments, the invention provides snake venom
protease polypeptides, e.g., a polypeptide: having the amino acid
sequence shown in SEQ ID NOs:2, 5, 8, 11, 14 or 17, or the amino
acid sequence encoded by the nucleic acid of SEQ ID Nos: 1, 3, 4,
6, 7, 9, 10, 12, 13, 15, 16, or 18; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NOs:2, 5, 8, 11, 14 or 17, or the amino acid sequence encoded by
the nucleic acid of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15,
16, or 18; or a sequence which has at least 85, 90, 95, 98 or 99%
sequence identity with, or which differs at no more than 1, 2, 5,
10, 15, or 20 residues from, one of the recited amino acid
sequences.
[0043] In other embodiments, the invention provides snake venom
protease light chain polypeptides, e.g., a polypeptide: having the
amino acid residues 41 to 179 (the numbering refers to the
consensus numbering in FIG. 23) of any of the amino acid sequences
shown in SEQ ID NOs:2, 5, 8, 11, 14 or 17, or the amino acid
residues 41 to 179 of the amino acid sequence encoded by the
nucleic acid of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16,
or 18; an amino acid sequence which is substantially identical to
amino acid residues 41 to 179 of the amino acid sequence shown in
SEQ ID NOs:2, 5, 8, 11, 14 or 17, or the amino acid sequence
encoded by the nucleic acid of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10,
12, 13, 15, 16, or 18; or a sequence which has at least 85, 90, 95,
98 or 99% sequence identity with, or which differs at no more than
1, 2, 5, 10, 15, or 20 residues from, one of the recited amino acid
sequences.
[0044] In other embodiments, the invention provides snake venom
protease heavy chain polypeptides, e.g., a polypeptide: having the
amino acid residues 235 to at least 453 of the amino acid sequence
shown in SEQ ID NOs:2, 5, 8, 11, 14 or 17, or the amino acid
residues 235 to at least 453 of the amino acid sequence encoded by
the nucleic acid of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15,
16, or 18; an amino acid sequence which is substantially identical
to amino acid residues 235 to at least 453 of the amino acid
sequence shown in SEQ ID NOs:2, 5, 8, 11, 14 or 17, or the amino
acid sequence encoded by the nucleic acid of SEQ ID Nos: 1, 3, 4,
6, 7, 9, 10, 12, 13, 15, 16, or 18; or a sequence which is at least
85, 90, 95, 98 or 99% sequence identity with, or which differs at
no more than 1, 2, 5, 10, 15, or 20 residues from, one of the
recited amino acid sequences.
[0045] In a related aspect, the invention further provides nucleic
acid constructs which include a snake venom protease nucleic acid
molecule described herein.
[0046] In a related aspect, the invention provides snake venom
protease polypeptides or fragments operatively linked to non-snake
venom protease polypeptides to form fusion proteins. In one
embodiment, the sequence encoding one or more or the light chain of
a snake venom protease, an activator polypeptide, and a heavy chain
venom protease can be linked to a sequence encoding a propeptide of
a non-snake venom prothrombin activating polypeptide, e.g., a human
factor Xa propeptide encoding sequence. In another embodiment, the
sequence encoding the light chain of a snake venom protease and the
sequence encoding the heavy chain of a snake venom protease can be
linked to each other by a nucleic acid sequence encoding an
activator peptide of a non-snake venom prothrombin activating
polypeptide, e.g., a human factor Xa activator peptide encoding
sequence. In other embodiments, an SVP sequence can be fused to a
sequence, preferably easily cleavable, which allows isolation,
e.g., fused to a GST moiety or to an epitope tag.
[0047] In another aspect, the invention features an isolated
protein comprising an amino acid sequence selected from any or all
of the group consisting of:
1 KREASLPDFVQS; [SEQ ID NO: 19] LKKSDNPSPDR; and [SEQ ID NO: 20]
SVX.sub.1VGEIX.sub.2X.sub.3SR. [SEQ ID NO: 21]
[0048] X.sub.1, X.sub.2 and X.sub.3 may be any amino acid.
[0049] Preferably, X.sub.1 is either valine or isoleucine, X.sub.2
is either asparginine or aspartic acid and X.sub.3 is either
arginine, lysine or isoleucine.
[0050] In one embodiment, the isolated protein further comprises an
amino acid sequence selected from the group consisting of:
2 MAPQLLLCLILTFLWSLPEAESNVFLKSK and [SEQ ID NO: 22] ANRFLQRTKR [SEQ
ID NO: 23]
[0051] In a particular embodiment, said prothrombin activating
protein of the invention is isolated from snake venom. Preferably,
said prothrombin activating protein of the invention is obtainable
from venom of an Australian snake selected from the non limiting
group consisting of: any brown snake (Psuedonaja sp.) including the
common brown snake (Pseudonaja textilis), taipan (Oxyuranus
scutellatus), mainland tiger (Notechis scutatus), rough scaled
(Tropidechis carinatus) and red-belly black snake (Pseudechis
porphyriacus).
[0052] In another aspect, the invention features an isolated
nucleic acid that encodes a snake venom protease polypeptide or
biologically active fragment thereof as described herein. In a
preferred embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NOs:2, 5, 8,
11, 14 or 17. In other embodiments, the invention provides isolated
nucleic acid molecules having the nucleotide sequence shown in SEQ
ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18, or a full
complement of SEQ ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or
18. In still other embodiments, the invention provides nucleic acid
molecules that are substantially identical (e.g., naturally
occurring allelic variants) to the nucleotide sequence shown in SEQ
ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18. In other
embodiments, the invention provides a nucleic acid molecule which
hybridizes under a stringency condition described herein to a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18, wherein the nucleic
acid encodes a full length snake venom protease polypeptide or an
active fragment thereof.
[0053] In a related aspect, the invention further provides nucleic
acid constructs that include a nucleic acid molecule encoding a
snake venom protease or portion thereof, e.g., as described herein.
In certain embodiments, the nucleic acid molecules of the invention
are operatively linked to native or heterologous regulatory
sequences. In other embodiments, the nucleic acid molecule includes
a nucleic acid encoding a propeptide, a nucleic acid sequence
encoding a light chain of a snake venom protease, a nucleic acid
sequence encoding an activator peptide, a nucleic acid sequence
encoding a heavy chain of a snake venom protease, wherein one or
more of the sequence encoding the propeptide and the sequence
encoding the activator peptide is not from a snake venom protease.
For example, one or more of the sequence encoding the propeptide
and activator peptide can be from a mammalian prothrombin
activator, e.g., a human prothrombin activator, e.g., human factor
Xa. Also included, are vectors and host cells containing the
nucleic acid molecules of the invention e.g., vectors and host
cells suitable for producing snake venom protease nucleic acid
molecules and polypeptides.
[0054] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection or amplification of snake venom protease-encoding nucleic
acids. For example, the invention includes primers spaced apart to
amplify: a full-length snake venom protease, e.g., a snake venom
protease described herein, or any domain or region of a snake venom
protease described herein.
[0055] In still another related aspect, isolated nucleic acid
molecules that are antisense to a snake venom protease-encoding
nucleic acid molecule are provided.
[0056] The invention also contemplates biologically active
fragments, variants, derivatives and homologs of the aforementioned
isolated proteins and nucleic acids of the invention.
[0057] In another aspect, the invention features an antibody that
binds an isolated snake venom protease polypeptide, e.g., a snake
venom protease polypeptide described herein. In one embodiment, the
antibody can bind to: the propeptide of a snake venom protease
polypeptide or fragments thereof described herein, a light chain of
a snake venom protease polypeptide or fragment thereof described
herein, an activator polypeptide of a snake venom protease
polypeptides or fragments thereof described herein, or a heavy
chain of a snake venom protease polypeptide or fragment thereof
described herein. In another embodiment, the antibody can bind a
portion of a snake venom protease which includes both the light and
heavy chains of a snake venom protease polypeptide described
herein. Antibodies can be used, e.g., to isolate snake venom
proteases from a sample.
[0058] In another aspect, the invention features a pharmaceutical
composition which includes an isolated snake venom protease
polypeptide or biologically active fragment thereof, e.g., an
isolated snake venom protease polypeptide described herein, and a
pharmaceutically acceptable carrier, diluent or excipient. In one
embodiment, the composition, e.g., pharmaceutical composition, has
a pH of about 5 to 9, preferably about 6.5 to 7. The composition,
e.g., pharmaceutical composition, can further include, e.g., a
stabilizer, such as a polyol. In such embodiments, the composition,
e.g., pharmaceutical composition can include about 5%, 10%, 20% or
more of a polyol (or polyols). An example of a polyol which can be
used in the composition is glycerol. In some embodiments, the
composition, e.g., pharmaceutical composition, does not include a
co-factor. In another embodiment, the composition, e.g.,
pharmaceutical composition can include one or more co-factors,
e.g., one or more of calcium, phospholipid and factor Va.
[0059] In another aspect, the invention provides methods of
screening for agents, e.g., compounds such as co-factors, that
modulate the activity of the snake venom polypeptides, e.g.,
compounds that modulate blood coagulation response and/or
processing of prothrombin to thrombin. In one embodiment, the
method can include providing a reaction mixture of prothrombin and
a snake venom protease, e.g., a snake venom protease described
herein, and contacting the reaction mixture with one or more
co-factors (e.g., one or more of calcium, a phospholipid, factor Va
and a vitamin, e.g., vitamin K). The reaction mixture can further
include, e.g., fibrinogen. The method can further include comparing
the activity of the snake venom protease on prothrombin processing
in the absence and presence of the agent, e.g., the co-factor. In
another embodiment, the method includes providing a sample (e.g., a
blood sample) and contacting the sample with a snake venom protease
in the absence and presence of an agent, e.g., a co-factor, and
comparing the effect of the co-factor on coagulation by the snake
venom protease. In yet another embodiment, the method can include
contacting platelets with a snake venom protease in the absence and
presence of an agent, e.g., a co-factor, to determine the effect of
the agent on platelet activation.
[0060] In one embodiment, the invention features a method of
measuring the level of activity by Citrate anticoagulated whole
blood or its plasma fraction that can be used to measure the
activity of the snake venom polypeptide (protease) by determining
the time for a solid clot to form. The measurement can be carried
out manually or by any of the automated coagulation measuring
devices. Furthermore, the activity of the protease can also be
measured by using tetrapeptides with a linked p-nitroanilide
(chromogenic substrates) which resemble specific domains of its
substrate (prothrombin). This assay is a simple calorimetric
measurement of rate of formation of p-nitroaniline in solution in a
substrate independent mixture.
[0061] In another aspect, the invention features a method of
treating a subject, e.g., by inducing haemostasis. The method
includes administering a snake venom protease of the invention to a
subject, thereby treating the subject, e.g., by inducing
haemostasis.
[0062] In a preferred embodiment, the subject is treated to inhibit
bleeding from a site on or in the subject's body. The treatment can
be used to inhibit bleeding which can occur in connection with
medical treatment, e.g., surgery. In other embodiments a wound,
trauma or other event is treated.
[0063] In some embodiments, the subject has a deficiency in the
ability to form or maintain a blood clot. This deficiency can be
due to a genetic defect or can be the result of medical treatment,
e.g., the administration of a drug which reduced the ability of the
subject to form or maintain a blood clot, e.g., coumadine or
Warfarin.
[0064] In one embodiment, the snake venom protease is administered
by a person other than the subject, while in other embodiments the
snake venom protease is self-administered. The person other than
the subject can be a health car provider but in some cases will not
be a health care provider. For example, in some embodiments, the
product will be used to treat battlefield trauma and will be
administered by a person other than a health care provider.
[0065] In some embodiments, the snake venom protease is provided to
the subject in advance of a need to use it, e.g., in the case of
subject has a deficiency in the ability to form or maintain a blood
clot or in the case of an individual who is believed to be at risk
for a traumatic wound, e.g., military personnel, persons working
with dangerous machinery, or generally those working in hazardous
occupations, such as farming or mining. The snake venom protease
can be supplied with written, recorded audio or video, or oral
instructions on its use.
[0066] In some embodiments the snake venom protease will be
provided in a form which allows the user (the subject or one who
administers it to the subject) to administer a measured dose. Thus,
the snake venom protease can be disposed in dispensing device,
e.g., a device which dispenses liquid, droplets, aerosols, dry
powder and the like, preferably in a metered dosage.
[0067] In another aspect, the invention provides a method of
activating prothrombin. The method includes contacting prothrombin
with a snake venom protease of the invention, to thereby activate
said prothrombin. The prothrombin can be activated in vitro or in
vivo. In one embodiment, the prothrombin can include
descarboxyprothrombin.
[0068] In particular embodiments, the pharmaceutical compositions
and methods of inducing haemostasis and/or prothrombin activation
can be used to prevent of blood loss from a wound. One such
embodiment, the composition may be that of a tissue sealant and/or
a fibrin glue. It is also contemplated that antifibrinolytic agents
may form part of such an embodiment. Anti fibrinolytic agents may
be selected from a non-limiting group including textilinin
(International Publication WO 99/58569), aprotinin and EACA, any of
which may be added to prevent lysis of the blood clot through the
inhibition of the action of plasmin or activators of plasmin.
[0069] In another aspect, the invention features a method of
obtaining a protein, nucleic acid, or library, or nucleic acid or
protein sequence information, e.g., as described herein. For
example obtaining a snake protein, e.g., an SVP, e.g., an SVP
described herein, or nucleic acid encoding a snake protein, e.g., a
nucleic acid encoding an SVP, e.g., an SVP described herein or any
of the libraries described herein. These are referred to herein as
"collection-based methods." The method includes: collecting an
Australian snake selected from the non-limiting group consisting of
a Pseudonaja textilis, Pseudonaja nuchalis, Pseudonaja affinis,
Pseudonaja inframacula, Oxyuranus scutellatus, Oxyuranus
microlepidotus, Notechis scutatus, Notechis ater niger, Notechis
ater serventyi, Hoplocephalus stephansii, Pseudechis porphiracus,
Australaps surperba, Tropedechis carinatus (or collecting tissue
from or produced by such a snake, e.g., eggs, or discarded tissue
such as a molted skin) and obtaining a protein, nucleic acid, or
library from the snake or from the progeny of the snake, or
obtaining sequence data from a protein or nucleic acid from the
snake, or from the progeny of the snake.
[0070] The method can include collecting a dead Australian snake or
capturing a live Australian snake or a live damaged Australian
snake. In one embodiment, the method further includes obtaining a
sample from the snake, e.g., obtaining a venom sample from the
snake, and obtaining the protein, or library of proteins, from the
sample, e.g., from the venom sample. Other embodiments include
obtaining a sample for the snake and obtaining a nucleic acid, or
library of nucleic acids, from the sample, e.g., from a venom
gland.
[0071] The method can further include determining a nucleic acid or
protein sequence from material taken form the snake or progeny
thereof.
[0072] The method can further include making a protein or nucleic
acid library from the collected snake or from progeny thereof.
[0073] The method can further include obtaining a polypeptide for
use, e.g., in animal, human or plant health, inductrial processing
or diagnostics.
[0074] In another embodiment, the method also includes collecting
the snake or sample and sending the snake or sample to a second
party, e.g., a party in another country to perform a subsequent
step of the method.
[0075] In another aspect the invention features a protein, nucleic
acid, or library, or nucleic acid or protein sequence information,
e.g., as described herein, which is made or produced by a method
described herein, e.g., one of the collection methods described
herein. In preferred embodiments the invention features a snake
protein, e.g., an SVP, e.g., an SVP described herein, or nucleic
acid encoding a snake protein, e.g., a nucleic acid encoding an
SVP, e.g., an SVP described herein or any of the libraries
described herein or the sequence information of any nucleic acid or
protein described herein made or produced by a method described
herein, e.g., a the collection methods described herein.
[0076] In one aspect, the invention features isolated polypeptides
comprising the sequence:
MAPQLLLCLILTFLWSLPEAESNVFLKSKX.sub.1ANRFLQRTKRX.-
sub.2NSLX.sub.3EEX.sub.4X.sub.5X.sub.6GNIERECIEEX.sub.7CSKEEAREX.sub.8FX.s-
ub.9DX.sub.10EKTEX.sub.11FWNVYVDGDQCSSNPCHYX.sub.12GX.sub.13CKDGIGSYTCTCLX-
.sub.14X.sub.15YEGKNCEX.sub.16X.sub.17LX.sub.18X.sub.19SCRX.sub.20X.sub.21-
NGNCWHFCK
X.sub.22VQX.sub.23X.sub.24X.sub.25QCSCAEX.sub.26YX.sub.27LGX.sub-
.28DGHSCVAX.sub.29GX.sub.30FSCGRNIKX.sub.31RNKRE
ASLPDFVQSX.sub.32X.sub.33-
AX.sub.34X.sub.35KKSDNPSPDIRIX.sub.36NGMDCKLGECPWQAX.sub.37LX.sub.38X.sub.-
39X.sub.40X.sub.41X.sub.42X.sub.43X.sub.44FCGGTILSPIX.sub.45VLTAAHCIX.sub.-
46X47X48X.sub.49X.sub.50X.sub.51SVX.sub.52VGEIX.sub.53X.sub.54SRX.sub.55X.-
sub.56X.sub.57X.sub.58X.sub.59LLSVDK.sub.60YVHX.sub.61KFVX.sub.62X.sub.63X-
.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70X.sub.71X.sub.72X.s-
ub.73X.sub.74X.sub.75X.sub.76X.sub.77YDYDIAIX.sub.78X.sub.79X.sub.80KTPIQF-
SENVVPACLPTADFAX.sub.81X.sub.82VLMKQD
X.sub.83GIX.sub.84SGFGX.sub.85X.sub.-
86X.sub.87X.sub.88X.sub.89X.sub.90X.sub.91X.sub.92SX.sub.93X.sub.94LKX.sub-
.95X.sub.96X.sub.97VPYVDRHTCMX.sub.98S
SX.sub.99X.sub.100X.sub.101ITX.sub.-
102X.sub.103MFCAGYDTLPX.sub.104DACQGDSGGPHITAYX.sub.105DTHFX.sub.106T
GIX.sub.107SWGEGCAX.sub.108X.sub.109GX.sub.110YGX.sub.111YTKX.sub.112SX.s-
ub.113FIX.sub.114WIK.sub.115X.sub.116MX.sub.117X.sub.118X.sub.119Z,
[0077] wherein X.sub.1, X.sub.10, X.sub.12-13, X.sub.15-16,
X.sub.19-23, X.sub.25, X.sub.27-30, X.sub.33-34, X.sub.37,
X.sub.39, X.sub.42-47, X.sub.50, X.sub.53-56, X.sub.58-62,
X.sub.64, X.sub.79, X.sub.81-83, X.sub.85-94, X.sub.96,
X.sub.99-105, X.sub.108-109, X.sub.113-115 and X.sub.117-119 are
each independently selected from any amino acid residue;
[0078] each of X.sub.2, X.sub.6, X.sub.11, X.sub.14, X.sub.26,
X.sub.31, X.sub.48X.sub.57 and X.sub.63 is a small amino acid
residue;
[0079] each of X.sub.3, X.sub.4, X.sub.8, X.sub.17, X.sub.18,
X.sub.35-36, X.sub.38, X.sub.51-52, X.sub.78, X.sub.80, X.sub.84,
X.sub.95, X.sub.98, X.sub.106-107, X.sub.111-112 and X.sub.116 is a
hydrophobic amino acid residue;
[0080] each of X.sub.5, X.sub.7 and X.sub.110 is a basic amino acid
residue;
[0081] each of X.sub.9, X.sub.40-41 and X.sub.49 is a charged amino
acid residue;
[0082] X.sub.24 is an acidic amino acid residue;
[0083] X.sub.32 is a neutral/polar amino acid residue;
[0084] X.sub.65-67, X.sub.70-72 and X.sub.75 are each independently
absent or selected from any amino acid residue;
[0085] X.sub.68 and X.sub.74 are each independently absent or
selected from acidic amino acid residues;
[0086] X.sub.69, X.sub.73 and X.sub.76 are each independently
absent or selected from hydrophobic amino acid residues;
[0087] X.sub.77 is absent or is a small amino acid residue; and
[0088] Z is absent or is a peptide of from 1-20 amino acids
[0089] In some embodiments, X.sub.1 is selected from a hydrophobic
or acidic amino acid residue, e.g., Val or a modified form thereof,
or Glu or a modified form thereof. In some embodiments, X.sub.2 is
selected from Ala or Ser or a modified form thereof. In some
embodiments, X.sub.3 is selected from Tyr or Phe or a modified form
thereof. In some embodiments, X.sub.4 is selected from Phe or Ile
or modified form thereof. In some embodiments, X.sub.5 is selected
from Lys or Arg or modified form thereof. In some embodiments,
X.sub.6 is selected from Pro or Ser or modified form thereof. In
some embodiments, X.sub.7 is selected from Arg or Lys or modified
form thereof In some embodiments, X.sub.8 is selected from Val or
Ile or modified form thereof In some embodiments, X.sub.9 is
selected from Glu or Lys or modified form thereof.
[0090] In some embodiments, X.sub.10 is a neutral/polar or acidic
amino acid residue, e.g., X.sub.10 is selected from Asp or Asn or
modified form thereof. In some embodiments, X.sub.11 is selected
from Thr or Ala or modified form thereof. In some embodiments,
X.sub.12 is a small or basic amino acid residue or modified form
thereof, e.g., X.sub.12 is selected from Gly or Arg or modified
form thereof. In some embodiments, X.sub.13 is a hydrophobic or
small amino acid residue or modified form thereof, e.g., X.sub.13
is selected from Ile or Thr or modified form thereof. In some
embodiments, X.sub.14 is selected from Pro or Ser or modified form
thereof. In some embodiments, X.sub.15 is a small or neutral/polar
amino acid residue, e.g., X.sub.15 is selected from Gly or Asn or
modified form thereof. In some embodiments, X.sub.16 is a basic or
neutral/polar amino acid residue, e.g., X.sub.16 is selected from
Arg, His or Lys or modified form thereof. In some embodiments,
X.sub.17 is selected from Val or Leu or modified form thereof. In
some embodiments, X.sub.18 is selected from Tyr or Phe or Leu or
modified form thereof. In some embodiments, X.sub.19 is a basic or
neural/polar amino acid residue, e.g., X.sub.19 is selected from
Lys or Gln or modified form thereof.
[0091] In some embodiments, X.sub.20 is a hydrophobic or small
amino acid residue, e.g., X.sub.20 is selected from Val, Phe or Ala
or modified form thereof. In some embodiments, X.sub.21 is an
acidic or hydrophobic amino acid residue, e.g., X.sub.21 is
selected from Asp or Phe or modified form thereof. In some
embodiments, X.sub.22 is a small or basic amino acid residue, e.g.,
X.sub.22 is selected from Pro, Asp or Phe or modified form thereof.
In some embodiments, X.sub.23 is a neutral/polar or small amino
acid residue, e.g., X.sub.23 is selected from Asn or Ser or
modified form thereof. In some embodiments, X.sub.24 is selected
from Asp or Glu or modified form thereof. In some embodiments,
X.sub.25 is a hydrophobic or small amino acid residue, e.g.,
X.sub.25 is selected from Ile or Thr or modified form thereof. In
some embodiments, X.sub.26 is selected from Gly or Ser or modified
form thereof. In some embodiments, X.sub.27 is a hydrophobic or
basic amino acid residue, e.g., X.sub.27 is selected from Leu or
Arg or modified form thereof. In some embodiments, X.sub.28 is an
acidic or hydrophobic amino acid residue, e.g., X.sub.28 is
selected from Glu, Asp or Val or modified form thereof. In some
embodiments, X.sub.29 is a small or acidic amino acid residue,
e.g., X.sub.29 is selected from Gly or Glu or modified form
thereof.
[0092] In some embodiments, X.sub.30 is a neutral/polar or acidic
amino acid residue, e.g., X.sub.30 is selected from Asn or Asp or
modified form thereof. In some embodiments, X.sub.31 is selected
from Thr or Ala or modified form thereof. In some embodiments,
X.sub.32 is selected from His or Gln or modified form thereof. In
some embodiments, X.sub.33 is a neutral/polar or basic amino acid
residue, e.g., X.sub.33 is selected from Asn or Lys or modified
form thereof. In some embodiments, X.sub.34 is a small or
hydrophobic amino acid residue, e.g., X.sub.34 is selected from Thr
or Ile or modified form thereof. In some embodiments, X.sub.35 is
selected from Leu or Val or modified form thereof. In some
embodiments, X.sub.36 is selected from Val or Ile or modified form
thereof. In some embodiments, X.sub.37 is a small or hydrophobic
amino acid residue, e.g., X.sub.37 is selected from Ala or Val or
modified form thereof. In some embodiments, X.sub.38 is selected
from Val, Leu or Ile or modified form thereof. In some embodiments,
X.sub.39 is an acidic or neutral/polar amino acid residue, e.g.,
X.sub.39 is selected from Asp or Asn or modified form thereof.
[0093] In some embodiments, X.sub.40 is selected from Asp, Glu or
Lys or modified form thereof. In some embodiments, X.sub.41 is
selected from Lys or Glu or modified form thereof. In some
embodiments, X.sub.42 is a charged or small amino acid residue,
e.g., X.sub.42 is selected from Lys, Glu or Gly or modified form
thereof. In some embodiments, X.sub.43 is a small or acidic amino
acid residue, e.g., X.sub.43 is selected from Gly, Asp or Glu or
modified form thereof. In some embodiments, X.sub.44 is a small or
hydrophobic amino acid residue, e.g., X.sub.44 is selected from Ala
or Val or modified form thereof. In some embodiments, X.sub.45 is a
hydrophobic or neutral/polar amino acid residue, e.g., X.sub.45 is
selected from Tyr or His or modified form thereof. In some
embodiments, X.sub.46 is a small or neutral/polar amino acid
residue, e.g., X.sub.46 is selected from Thr or Asn or modified
form thereof. In some embodiments, X.sub.47 is an acidic or
neutral/polar amino acid residue, e.g., X.sub.47 is selected from
Glu or Gln or modified form thereof. In some embodiments, X.sub.48
is selected from Thr or Ser or modified form thereof. In some
embodiments, X.sub.49 is selected from Glu or Lys or modified form
thereof.
[0094] In some embodiments, X.sub.50 is a small, hydrophobic or
neutral/polar amino acid residue, e.g., X.sub.50 is selected from
Thr, Met, His or Ser or modified form thereof. In some embodiments,
X.sub.51 is selected from Ile or Val or modified form thereof. In
some embodiments, X.sub.52 is selected from Val or Ile or modified
form thereof. In some embodiments, X.sub.53 is an acidic or
neutral/polar amino acid residue, e.g., X.sub.53 is selected from
Asp or Asn or modified form thereof. In some embodiments, X.sub.54
is a basic or hydrophobic amino acid residue, e.g., X.sub.54 is
selected from Arg or Ile or modified form thereof. In some
embodiments, X.sub.55 is a small or basic amino acid residue, e.g.,
X.sub.55 is selected from Ala or Lys or modified form thereof. In
some embodiments, X.sub.56 is an acidic or neutral/polar amino acid
residue, e.g., X.sub.56 is selected from Glu or Asn or modified
form thereof. In some embodiments, X.sub.57 is selected from Pro or
Thr or modified form thereof. In some embodiments, X.sub.58 is a
small or basic amino acid residue, e.g., X.sub.58 is selected from
Gly or Arg or modified form thereof. In some embodiments, X.sub.59
is a small, basic or neutral/polar amino acid residue, e.g.,
X.sub.59 is selected from Pro, Arg or His or modified form
thereof.
[0095] In some embodiments, X.sub.60 is a hydrophobic or small
amino acid residue, e.g., X.sub.60 is selected from Val, Ile or Ala
or modified form thereof. In some embodiments, X.sub.61 is a basic,
neutral/polar or small amino acid residue, e.g., X.sub.61 is
selected from Lys, Gin or Thr or modified form thereof. In some
embodiments, X.sub.62 is a small or hydrophobic amino acid residue
e.g., X.sub.62 is selected from Pro or Leu or modified form
thereof. In some embodiments, X.sub.63 is selected from Pro or Ala
or modified form thereof. In some embodiments, X.sub.64 is a basic,
small or neutral/polar amino acid residue e.g., X.sub.64 is
selected from Lys, Thr or Asn or modified form thereof. In some
embodiments, X.sub.65 when present is a basic, small or hydrophobic
amino acid residue e.g., X.sub.65 is selected from Lys, Ser or Tyr
or modified form thereof. In some embodiments, X.sub.66 when
present is a small or hydrophobic amino acid residue, e.g.,
X.sub.66 is selected from Ser, Gly or Tyr or modified form thereof.
In some embodiments, X.sub.67 when present is a neutral/polar or
hydrophobic amino acid residue, e.g., X.sub.67 is selected from Gln
or Tyr or modified form thereof. In some embodiments, X.sub.68 when
present is Glu or modified form thereof. In some embodiments,
X.sub.69 when present is selected from Phe or Val or modified form
thereof.
[0096] In some embodiments, X.sub.70 when present is a hydrophobic
or neutral/polar amino acid residue, e.g., X.sub.70 is selected
from Tyr or His or modified form thereof. In some embodiments,
X.sub.71 when present is an acidic or neutral/polar amino acid
residue, e.g., X.sub.71 is selected from Glu or Gln or modified
form thereof. In some embodiments, X.sub.72 when present is a basic
or neutral/polar amino acid residue, e.g., X.sub.72 is selected
from Lys or Asn or modified form thereof. In some embodiments,
X.sub.73 when present is selected from Phe or Ile or modified form
thereof. In some embodiments, X.sub.74 when present is Asp or
modified form thereof. In some embodiments, X.sub.75 when present
is a hydrophobic or basic amino acid residue, e.g., X.sub.75 is
selected from Leu or Arg or modified form thereof. In some
embodiments, X.sub.76 when present is selected from Val or Phe or
modified form thereof. In some embodiments, X.sub.77 when present
is selected from Ser or Ala or modified form thereof. In some
embodiments, X.sub.78 is selected from Ile or Leu or modified form
thereof. In some embodiments, X.sub.79 is a neutral/polar or basic
amino acid residue, e.g., X.sub.79 is selected from Gln or Arg or
modified form thereof.
[0097] In some embodiments, X.sub.80 is selected from Met or Leu or
modified form thereof. In some embodiments, X.sub.81 is a
neutral/polar or basic amino acid residue, e.g., X.sub.81 is
selected from Asn or Lys or modified form thereof. In some
embodiments, X.sub.82 is a neutral/polar or acidic amino acid
residue, e.g., X.sub.82 is selected from Gln or Glu or modified
form thereof. In some embodiments, X.sub.83 is a hydrophobic or
small amino acid residue, e.g., X.sub.83 is selected from Phe or
Ser or modified form thereof. In some embodiments, X.sub.84 is
selected from Val or Ile or modified form thereof In some
embodiments, X.sub.85 is a small, basic or neutral/polar amino acid
residue, e.g., X.sub.85 is selected from Gly, Arg or His or
modified form thereof. In some embodiments, X.sub.86 is a
hydrophobic or small amino acid residue e.g., X.sub.86 is selected
from Ile or Thr or modified form thereof. In some embodiments,
X.sub.87 is a hydrophobic, basic or neutral/polar amino acid
residue, e.g., X.sub.87 is selected from Phe, Arg or Gln or
modified form thereof. In some embodiments, X.sub.88 is an acidic,
small or hydrophobic amino acid residue, e.g., X.sub.88 is selected
from Glu, Ser or Phe or modified form thereof. In some embodiments,
wherein X.sub.89 is a basic, small or hydrophobic amino acid
residue, e.g., X.sub.89 is selected from Arg, Lys, Gly, or Ile or
modified form thereof.
[0098] In some embodiments, X.sub.90 is a small or neutral/polar
amino acid residue, e.g., X.sub.90 is selected from Gly, or Gln or
modified form thereof. In some embodiments, X.sub.91 is a small,
neutral/polar or hydrophobic amino acid residue, e.g., X.sub.91 is
selected from Pro, Gln or Tyr or modified form thereof. In some
embodiments, X.sub.92 is a neutral/polar or small amino acid
residue, e.g., X.sub.92 is selected from Asn, Gln or Thr or
modified form thereof. In some embodiments, X.sub.93 is a basic or
neutral/polar amino acid residue, e.g., X.sub.93 is selected from
Lys or Asn or modified form thereof. In some embodiments, X.sub.94
is a small or hydrophobic amino acid residue e.g., X.sub.94 is
selected from Thr or Ile or modified form thereof. In some
embodiments, X.sub.95 is selected from Leu, Val or Ile or modified
form thereof. In some embodiments, X.sub.96 is a basic or small
amino acid residue, e.g., X.sub.96 is selected from Lys or Thr or
modified form thereof. In some embodiments, X.sub.97 is selected
from Val or Ile or modified form thereof. In some embodiments,
X.sub.98 is selected from Leu or Val or modified form thereof. In
some embodiments, X.sub.99 is a neutral/polar or acidic amino acid
residue, e.g., X.sub.99 is selected from Asn, Glu or Asp or
modified form thereof.
[0099] In some embodiments, X.sub.100 is a hydrophobic or small
amino acid residue, e.g., X.sub.100 is selected from Phe or Ser or
modified form thereof. In some embodiments, X.sub.101 is a small or
basic amino acid residue, e.g., X.sub.101 is selected from Pro or
Arg or modified form thereof. In some embodiments, X.sub.102 is a
small or neutral/polar amino acid residue, e.g., X.sub.102 is
selected from Pro or Gln or modified form thereof. In some
embodiments, X.sub.103 is a small or neutral/polar amino acid
residue, e.g., X.sub.103 is selected from Thr or Asn or modified
form thereof. In some embodiments, X.sub.104 is a neutral/polar or
basic amino acid residue, e.g., X.sub.104 is selected from Gln or
Arg or modified form thereof. In some embodiments, X.sub.105 is a
basic or small amino acid residue, e.g., X.sub.105 is selected from
Arg or Gly or modified form thereof. In some embodiments, X.sub.106
is selected from Ile or Val or modified form thereof. In some
embodiments, X.sub.107 is selected from Val or Ile or modified form
thereof. In some embodiments, X.sub.108 is a basic or neutral/polar
amino acid residue, e.g., X.sub.108 is selected from Arg, Gln or
Lys or modified form thereof. In some embodiments, X.sub.109 is a
basic or small amino acid residue, e.g., X.sub.109 is selected from
Lys or Thr or modified form thereof.
[0100] In some embodiments, X.sub.110 is selected from Arg or Lys
or modified form thereof. In some embodiments, X.sub.111 is
selected from Ile or Val or modified form thereof. In some
embodiments, X.sub.112 is selected from Leu or Val or modified form
thereof. In some embodiments, X.sub.113 is a basic or neutral/polar
amino acid residue, e.g., X.sub.113 is selected from Lys or Asn or
modified form thereof. In some embodiments, X.sub.114 is a small or
hydrophobic amino acid residue, e.g., X.sub.114 is selected from
Pro or Leu or modified form thereof. In some embodiments, X.sub.115
is a basic or small amino acid residue, e.g., X.sub.115 is selected
from Arg, Lys or Ala or modified form thereof. In some embodiments,
X.sub.116 is selected from Ile or Val or modified form thereof. In
some embodiments, X.sub.117 a basic or small amino acid residue,
e.g., X.sub.117 is selected from Arg or Ser or modified form
thereof. In some embodiments, X.sub.118 is a neutral/polar, basic
or hydrophobic amino acid residue, e.g., X.sub.118 is selected from
Gln, Lys or Leu or modified form thereof. In some embodiments,
X.sub.119 is a basic or neutral/polar amino acid residue, e.g.,
X.sub.119 is selected from Lys or His or modified form thereof.
[0101] In some embodiments, Z is present and comprises the sequence
X.sub.118PSTESSTGRL, wherein X.sub.118 is any amino acid residue.
In some embodiments, X.sub.118 is a hydrophobic or neutral polar
amino acid residue, e.g., X.sub.118 is selected from Leu or Gln or
modified form thereof.
[0102] In some embodiments, X.sub.65-77 represents a sequence of n
amino acids where n is from 0 to 13 amino acid residues, e.g., the
sequence is selected from KX.sub.119X.sub.120EFYEKFDLVS, SYYQNIDRFA
or YYYVHQNFDRVA, wherein X.sub.119 is a small amino acid residue,
e.g., X.sub.119 is selected from Ser or Gly or modified form
thereof; and X.sub.120 is any amino acid residue, e.g., X.sub.120
is selected from Gln or Tyr or modified form thereof.
[0103] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE TABLES AND FIGURES
[0104] Table 1: Characterization of samples during purification of
the Brown snake venom protease using Sephacryl S-300.
[0105] Table 2: Characterization of samples during purification of
Brown snake venom protease using Superdex 200.
[0106] Table 3: Characterization of samples during purification of
Brown snake venom protease, protocol 1.
[0107] Table 4: Characterization of samples during purification of
Brown snake venom protease, protocol 2.
[0108] Table 5: Characterization of samples during purification of
Brown snake venom protease, protocol 3.
[0109] Table 6: Characterization of samples during purification of
Brown snake venom protease, protocol 4.
[0110] Table 7: Hydrolysis of S-2222 by Brown snake venom protease
complex with and without accessory components (Brown snake venom
protease complex alone, Brown snake venom protease complex with 10
mM CaCl.sub.2 and Brown snake venom protease complex with 10 mM
CaCl.sub.2 and phospholipid).
[0111] Table 8: Clotting time of citrated plasma by Brown snake
venom protease complex alone, Brown snake venom protease complex
with 10 mM CaCl.sub.2 and Brown snake venom protease complex with
10 mM CaCl.sub.2 and phospholipid.
[0112] Table 9: Clotting time of citrated plasma clotting
assays.+-.Ca.sup.2+, with added isolated snake venom protease
derived from P. textilis (Brown snake).
[0113] Table 10: Clotting of citrated plasma by Brown snake venom
protease.
[0114] Table 11: Initial rates of hydrolysis of S-2222 by isolated
snake venom protease derived from P. textilis, with or without
added 10 mM Ca.sup.2+.
[0115] Table 12: Approximate clotting times of clots produced in
human citrated plasma using Brown snake venom protease with and
without 40 mM CaCl.sub.2, and with 40 CaCl.sub.2 alone.
[0116] Table 13: Determination of the molecular mass of Brown snake
venom protease by various methods.
[0117] Table 14: Blood loss in a mouse tail-vein bleeding model
treated with Brown snake venom protease.
[0118] Table 15: Blood loss from Brown snake venom protease (test)
and saline (control) treated mice. Data for each individual test
mouse can be seen and also average blood loss.+-.standard deviation
(SD).
[0119] Table 16: Clotting of citrated human plasma by various
Australian and exotic snake venoms.
[0120] FIG. 1: Elution profile after chromatography of P. textilis
venom (10 mL; 233 mg) on a column (2.5.times.16 cm) of
ConA-Sepharose 4B in 0.05 M Tris-HCl, pH 7.4. A. Trace of
chromatography pattern. The eluting buffer (0.02 M methyl .alpha.-D
mannopyranoside in 0.05 M Tris-HCl) was applied to the column at
arrow B. Fractions with S-2222 hydrolytic activity were pooled and
concentrated (designated by the line at A).
[0121] FIG. 2: SDS PAGE of pooled and concentrated peak from
ConA-Sepharose 4B chromatography. Lane 1. Molecular weight markers
(sizes are shown in kDa). Lane 2. Brown snake venom protease
complex without .beta.-mercaptoethanol. Lane 3. Brown snake venom
protease complex with .beta.-mercaptoethanol.
[0122] FIG. 3: The effect on citrated plasma clotting time and
hydrolysis of S-2222 by snake venom protease complex derived from
P. textilis treated with 0.8 M NaSCN.
[0123] FIG. 4: HPLC data of Brown snake venom serine protease.
[0124] FIG. 5: SDS PAGE.+-..beta.-Me. Lane 1--10 .mu.l BIO-RAD
marker, Lane 2--20 .mu.i P. textilis venom, Lane 3--20 .mu.l intact
Pt-PA, Lane 4--20 .mu.l Sephacryl S-300 (1) pooled fractions 30-43,
Lane 5--20 .mu.l Sephacryl S-300 (2) pooled fractions 25-29, Lane
6, 7 and 8--10 .mu.l Sephacryl S-300 (3) pooled fractions 25-29,
Lane 9--20 .mu.l Sephacryl S-300 (3) pooled fractions
25-29+.beta.-Me and Lane 10--20 .mu.l intact serum venom protease
complex +.beta.-Me.
[0125] FIG. 6: SDS-PAGE of Brown snake venom serine protease, with
or without .beta.-Me. Lane 1--BIO-RAD marker, Lane 2--whole P.
textilis venom, Lane 3--Sephacryl S-300 (3) pooled fractions 30-43,
Lane 4--Sephacryl S-300 (#3), Lane 5--S-300 (#3)+.beta.-Me, Lane
6--S300 (#3), Lane 7--Sephacryl S-300 (#3)+.beta.-Me, Lane
8--Sephacryl S-300 (3) pooled fractions 30-43+.beta.-Me, Lane
9--intact Brown snake venom protease complex+.beta.-Me and Lane
10--BIO-RAD marker. # represent the pooled and concentrated active
peak from Sephacryl S-300 chromatographies of Brown snake venom
protease complex as above. All samples consisted of 10 .mu.l
aliquots.
[0126] FIG. 7A: Elution profile after chromatography step 1 of
Brown snake venom protease complex (18 mL; 50.4 mg) on a column
(2.5.times.90 cm) of Superdex 200 in 0.05 M Tris-HCl, pH 7.4 with
0.8 M NaSCN. Fractions with S-2222 activity were pooled and
concentrated, designated by the line at A.
[0127] FIG. 7B: Chromatography step 2 as per conditions of FIG.
10A.
[0128] FIG. 7C: SDS PAGE of samples from purification of Brown
snake venom protease with Superdex 200. Lanes 1 & 2. Pooled
concentrate from chromatography step 1 with (2) and without (1)
.beta.-mercaptoethanol. Lanes 3 & 4. Pooled concentrate from
chromatography step 2 with (5) and without (4) -mercaptoethanol.
Lane 5. Molecular weight markers (sizes are shown in kDa). Arrows
A, B and C indicate impurities in lane 4.
[0129] FIG. 8A: Clotting of citrated plasma by Brown snake venom
protease (referred to as Pt-PA protease) without accessory
components (data points are means of duplicate measurements).
[0130] FIG. 8B: Clotting of citrated plasma by Brown snake venom
("Pt-PA") protease with 10 mM CaCl.sub.2.
[0131] FIG. 8C: Clotting of citrated plasma by Brown snake venom
("Pt-PA") protease with 10 mM CaCl.sub.2 and phospholipid.
[0132] FIG. 9A: Hydrolysis of S-2222 by Brown snake venom protease
(referred to as Pt-PA protease) without accessory components (data
points are means of duplicate measurements).
[0133] FIG. 9B: Hydrolysis of S-2222 by Brown snake venom protease
without accessory components (data points are means of duplicate
measurements) with 10 mM CaCl.sub.2.
[0134] FIG. 9C: Hydrolysis of S-2222 by Brown snake venom protease
without accessory components (data points are means of duplicate
measurements) with 10 mM CaCl.sub.2.
[0135] FIG. 9D: Slope and R.sub.2 value of respective plots in
FIGS. 12A, 12B and 12C. The R.sub.2 value is the correlation
coefficient for a straight line.
[0136] FIG. 10: Prothrombin activation by Brown snake venom
protease. Prothrombin (100 .mu.L of a 1.3 mg/mL preparation) was
converted to thrombin by Brown snake venom protease (20 .mu.L of a
1.3 mg/mL preparation) in a total volume of 500 .mu.L for time
periods indicated on the X-axis. An aliquot of each reaction was
then added to a citrated plasma clotting assay and clotting times
measured (Y-axis).
[0137] FIG. 11A: SDS PAGE without reduction of prothrombin after
incubation with Brown snake venom protease. Brown snake venom
protease was added to prothrombin at 0 min (time, t=0); Lane 1,
molecular weight markers (sizes shown in kDa); Lane 2, t=0; Lane 3,
t=6 min; Lane 4, t=24 h; Lane 5, t=48 h. PT, prothrombin; PT.sub.1,
prethrombin 1; T, thrombin; F.sub.1.2, fragment 1.2; PT.sub.2,
prethrombin 2; F.sub.1, fragment 1.
[0138] FIG. 11B: Hydrolysis of S-2238 by Brown snake venom
protease-generated thrombin.
[0139] FIG. 12: Proposed model of prothrombin activation by Brown
snake venom protease. Arrows indicate bonds that are cleaved by
thrombin and Brown snake venom protease.
[0140] FIG. 13: SDS PAGE of fibrin clots in the presence of
.beta.-mercaptoethanol. Lane 1. Molecular weight markers (sizes are
shown in kDa). Lane 2. Fibrin clot obtained by the action of 22
.mu.g Brown snake venom protease alone on citrated plasma. Lane 3.
Fibrin clot obtained by the action of 22 .mu.g Brown snake venom
protease with 40 mM CaCl.sub.2 on citrated plasma. Lane 4. Fibrin
clot produced with 40 mM CaCl.sub.2. Lane 5. Human fibrinogen. The
Greek symbols on the right hand side of the gel are indicative of
the chains of human fibrinogen including A.alpha. (.alpha. monomer
and fibrinopeptide A), B.beta. (.beta. monomer with fibrinopeptide
B) and .gamma. chains.
[0141] FIG. 14: Mapping of protease active site. SDS PAGE of
purified Brown snake venom protease with and without DNS-GGACK
treatment. Lanes 1 and 2. Brown snake venom protease complex
inhibited with DNS-GGACK with (2) and without
.beta.-mercaptoethanol (1). Lanes 3 and 4. Brown snake venom
protease inhibited with DNS-GGACK with (4) and without
.beta.-mercaptoethanol (3). Lanes 5-8 are a repeat of lanes 1-4
without DNS-GGACK and stained with Coomassie blue. Lane 9.
Molecular weight markers (sizes are shown in kDa).
[0142] FIG. 15: Amino acid sequence alignment of a protein fragment
of Brown snake venom protease, trocarin and human factor Xa
comprising a putative active site having proposed interacting
histidines shown in bold.
[0143] FIG. 16: Amino acid sequence alignment of part of the
predicted Brown snake venom protease heavy chain and Trocarin. An
Expect (E) value is a parameter depicting the number of hits
expected by chance when performing a search in the NCBI database.
The closer the E value to zero, the more significant the sequence
match. The E value decreases exponentially with Score given to a
match between two sequences and also depends on the length of
sequences compared. An Expect value of 1 means that within the
database one match is expected a similar score by chance.
Score=39.7, Expect=0.004; Identities=11/11 (100%), Positives=11/11
(100%)
[0144] FIG. 17: Amino acid sequence alignment of a part of the
predicted Brown snake venom protease heavy chain and human factor
Xa.
[0145] FIG. 18: Amino acid sequence alignment of a part of the
predicted Brown snake venom protease light chain and Trocarin.
[0146] FIG. 19: Sequence alignment of a part of the predicted Brown
snake venom protease light chain and mouse factor X. Score=24.8,
Expect=116; Identities=9/12 (75%), Positives=9/12 (76%).
[0147] FIG. 20A: Nucleotide acid sequence [SEQ ID NO: 1] encoding
snake venom protease of P. textilis (common brown snake).
[0148] FIG. 20B: Amino acid sequence [SEQ ID NO: 2] of snake venom
protease of P. textilis (common brown snake).
[0149] FIG. 21. Amino acid sequence alignment between snake venom
proteases isolated from venom glands of the following Australian
snakes: P. textilis (brown) [SEQ ID NO: 2], O. scutellatus (coastal
taipan) [SEQ ID NO: 5], P. porphyriacus (red-belly black) [SEQ ID
NO: 11], N. scutatus (mainland tiger) [SEQ ID NO: 14], T. carinatus
(rough scale) [SEQ ID NO: 17] and Trocarin [SEQ ID NO: 31].
[0150] FIG. 22. Amino acid sequence alignment of isolated snake
venom proteases with human Xa [SEQ ID NO: 27]. Shown are amino acid
sequences of snake venom proteases derived from the following
snakes: brown [SEQ ID NO: 2], Coastal Taipan [SEQ ID NO: 5], Red
Belly [SEQ ID NO: 11], Rough scale "Roughie" [SEQ ID NO: 14] and
Mainland Tiger [SEQ ID NO: 17].
[0151] FIG. 23. Amino acid sequence alignment between snake venom
proteases isolated from venom glands of the Australian snakes P.
textilis (brown) [SEQ ID NO: 2], O. scutellatus (coastal taipan)
[SEQ ID NO: 5], O. microepidotus (inland taipan) [SEQ ID NO:8], P.
porphyriacus (red-belly black) [SEQ ID NO: 11], N. scutatus
(mainland tiger) [SEQ ID NO: 14], and T. carinatus (rough scale)
[SEQ ID NO: 17].
[0152] FIG. 24 Amino acid sequence alignment between snake venom
proteases isolated from venom glands of the Australian snakes P.
textilis (brown) [SEQ ID NO: 2], O. scutellatus (coastal taipan)
[SEQ ID NO: 5], O. microepidotus (inland taipan) [SEQ ID NO:8], P.
porphyriacus (red-belly black) [SEQ ID NO: 11], N. scutatus
(mainland tiger) [SEQ ID NO: 14], T. carinatus (rough scale) [SEQ
ID NO: 17] and a consensus sequence [SEQ ID NO: ].
[0153] FIG. 25. Nucleotide sequence alignment of nucleic acids
encoding snake venom proteases derived from following Australian
snakes: P. textilis (brown) [SEQ ID NO: 1], O. scutellatus (costal
taipan) [SEQ ID NO: 4], O. microlepidotus (inland taipan),[SEQ ID
NO:7], P. porphyriacus (red-belly black) [SEQ ID NO: 10], N.
scutatus (mainland tiger) [SEQ ID NO: 13], and T. carinatus (rough
scale) [SEQ ID NO: 16].
[0154] FIG. 26. Nucleotide sequence alignment of nucleic acids
encoding snake venom proteases derived from following Australian
snakes: P. textilis (brown) [SEQ ID NO: 1], O. scutellatus (costal
taipan) [SEQ ID NO: 4], P. porphyriacus (red-belly black) [SEQ ID
NO: 10], N. scutatus (mainland tiger) [SEQ ID NO: 13], T. carinatus
(rough scale) [SEQ ID NO: 16] and human Factor Xa [SEQ ID NO:
26].
[0155] FIG. 27: Shows mouse tails with and without treatment with
Brown snake venom protease (note the large clot formed with
protease treatment).
[0156] FIG. 28: Box plot of mouse bleeding results. Each box
represents a range that comprises 50% of values. The whiskers are
lines that extend from the box to the highest and lowest values.
The line across the box indicates the median.
DETAILED DESCRIPTION OF THE INVENTION
[0157] Snake venoms are an abundant source of proteins and other
constituents that affect the haemostatic mechanism of mammals via
inhibition and/or activation of factors within the pathways of
platelet aggregation, fibrinolysis and the coagulation cascade. Of
particular note are the snake venom proteases unique to Australian
elapid snake species. Normally, proteolytic cleavage of prothrombin
to its active from thrombin, is catalysed by the prothrombinase
complex in mammalian systems. The functional protease within
prothrombinase is factor Xa. However, for optimal activity, the Xa
enzyme requires factor Va as a cofactor in the presence of calcium
ions and phospholipids.
[0158] The invention is based, in part, from the isolation of snake
venom proteases from venom of Australian snakes. Examples of
Australian snakes include the Australian common brown snake
Pseudonaja textilis, coastal taipan (Oxyuranus scutellatus), inland
taipan (Oxyuranus microlepidotus), mainland tiger (Notechis
scutatus), rough scaled (Tropidechis carinatus) and red-belly black
snake (Pseudechis porphyriacus) and other snakes from the genus
Elapidae. The snake venom proteases of the invention mimic the
effect of factor Xa in vivo, cleaving prothrombin to thrombin,
however they do so in the absence of cofactors, such as factor Va,
phospholipid and calcium ions. Thus, the snake venom proteases
described herein act as either complete or partially complete
prothrombin activators. The term "complete prothrombin activator"
as used herein refers to a snake venom protease which process
prothrombin to thrombin in the absence of calcium, phospholipids
and factor Va. Examples of snake venom proteases which act as
complete prothrombin activators include snake venom proteases from
the brown snake and the taipan snakes. The term "partially complete
prothrombin activators" as used herein refers to snake venom
proteases which process prothrombin to thrombin in the absence of
calcium and phospholipids, but do require the presence of factor
Va.
[0159] In one particular embodiment, the invention provides
isolated snake venom proteases isolated from the venom of the
common Australian brown snake (P. textilis), taipan (Oxyuranus
scutellatus)-coastal or inland, mainland tiger (Notechis scutatus),
rough scaled (Tropidechis carinatus) and red-belly black snake
(Pseudechis porphyriacus).
[0160] A snake venom protease of the invention may be isolated from
a prothrombinase complex referred to herein as a "Snake venom
protease complex" The snake venom protease complex may comprise
several proteins and/or cofactors. Snake venom proteases of the
invention include, for example, those proteins shown in FIG. 23 and
proteolytically digested sub-fragments thereof. FIG. 23 depicts the
amino acid sequence of a snake venom protease from brown snake (SEQ
ID NO:2); the amino acid sequence of a snake venom protease from
coastal taipan snake (SEQ ID NO:5); the amino acid sequence of a
snake venom protease from inland taipan snake (SEQ ID NO:8); the
amino acid sequence of a snake venom protease from red belly black
snake (SEQ ID NO:11); the amino acid sequence of a snake venom
protease from tiger snake (SEQ ID NO: 14); and the amino acid
sequence of a snake venom protease from rough scale snake (SEQ ID
NO:17).
[0161] The snake venom proteases of the invention contain a
significant number of structural characteristics in common with
each other. The term "family" when referring to the protein and
nucleic acid molecules of the invention means two or more proteins
or nucleic acid molecules having a common structural domain or
motif and having sufficient amino acid or nucleotide sequence
homology as defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. Members of a family can also have common
functional characteristics.
[0162] As used herein, a "snake venom protease activity",
"biological activity of a snake venom protease" or "functional
activity of a snake venom protease", refers to an activity exerted
by a snake venom protease protein, polypeptide or nucleic acid
molecule. For example, a snake venom protease activity can be one
or more of: the ability to process prothrombin to thrombin (e.g.,
the ability to cleave prothrombin between the arginine residue 274
and the threonine residue 275 of prothrombin and between the
arginine residue 323 and the isoleucine residue 324 of prothrombin,
e.g., the ability to cleave prothrombin between the arginine
residue 274 and the threonine residue 275 of prothrombin and
between the arginine residue 323 and the isoleucine residue 324 of
prothrombin but not to cleave prothrombin between the arginine
residue 155 and the serine residue 156 and/or between the arginine
residue 286 and the threonine residue 287); the ability to produce
clotting in citrate-treated plasma; the ability to process
prothrombin and/or produce clotting in the absence of calcium and
phospholipid. The isolated snake venom proteases of the invention
are characterized by having a prothrombinase activity largely
independent of calcium as shown, for example, in Tables 8-12.
[0163] The invention features snake venom polypeptides and
biologically active fragments thereof, that are complete or
partially complete prothrombin activators. A complete or partial
activator shows significantly greater activity in the absence of
cofactors than does an incomplete activator, e.g., human factor X
or trocarin. Embodiments of complete or partially complete
activators of the invention have a level of activity that is about
0.4% of the activity of the complete prothrombin activator in
combination with Ca.sup.2+ and phospholipids. The activity of the
complete or partially complete prothrombin activator alone in
preferred embodiments is at least 1.5, 2, 4, 10, 15, 20, 50, 100,
1000, or 4000 fold (two to four orders of magnitude) higher than
that of an incomplete activator, e.g., human factor Xa, or
trocarin, alone. This comparison is made between a snake venom
protease and an incomplete activator measured under the same or
similar conditions, e.g., in the absence of Ca and phospholipids.
In preferred embodiments, the % of activity (i.e., the activity of
the complete or partially complete activator in the absence of Ca
and phospholipid as a % of that seen with the same activator in the
presence of Ca and phospholipids) of a complete or partially
complete is at least 1.5, 2, 4, 10, 15, 20, 50, 100, 1000, or 4000
fold greater than the same % shown by an incomplete activator,
e.g., human factor X or trocarin. Preferred complete or partially
complete activators will clot citrated plasma at concentration of
about 10.sup.-10 to 10.sup.-06 M, e.g., at 10.sup.-8 or 10.sup.-7
M, giving clotting times of about 50 to 15 seconds, demonstrating
Ca.sup.2+ and phospholipid independence. Accordingly, the
prothrombin activator shows kinetic properties of cofactor
independence (calcium ions and/or phospholipid) in the
concentration range of about 10.sup.-10 to 10.sup.-06 M
concentration range being a suitable working range to reduce blood
loss.
[0164] The snake venom protease proteins, fragments thereof, and
derivatives and other variants of the sequence in SEQ ID NO:2, 5,
8, 11, 14 and 17, are collectively referred to as "polypeptides or
proteins of the invention" or "snake venom protease polypeptides or
proteins". Nucleic acid molecules encoding such polypeptides or
proteins are collectively referred to as "nucleic acids of the
invention" or "snake venom protease-encoding nucleic acids." Snake
venom protease molecules refer to snake venom protease nucleic
acids, polypeptides, and antibodies.
[0165] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g.,
an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be
synthesized from nucleotide analogs. The nucleic acid molecule can
be single-stranded or double-stranded, but preferably is
double-stranded DNA. FIG. 26 depicts a nucleic acid sequence
encoding a snake venom protease from brown snake (SEQ ID NO:1,
coding region SEQ ID NO:3); a nucleic acid sequence encoding a
snake venom protease from coastal taipan snake (SEQ ID NO:4, coding
region SEQ ID NO:6); a nucleic acid sequence encoding a snake venom
protease from inland taipan snake (SEQ ID NO:7), coding region SEQ
ID NO:9); a nucleic acid sequence encoding a snake venom protease
from red belly black snake (SEQ ID NO:10, coding region SEQ ID
NO:12); a nucleic acid sequence encoding a snake venom protease
from tiger snake (SEQ ID NO: 13, coding region SEQ ID NO:15); and a
nucleic acid sequence encoding a snake venom protease from rough
scale snake (SEQ ID NO:16, coding region SEQ ID NO:18).
[0166] The term "isolated nucleic acid molecule" or "purified
nucleic acid molecule" includes nucleic acid molecules that are
separated from other nucleic acid molecules present in the natural
source of the nucleic acid. For example, with regards to genomic
DNA, the term "isolated" includes nucleic acid molecules which are
separated from the chromosome with which the genomic DNA is
naturally associated. Preferably, an "isolated" nucleic acid is
free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and/or 3' ends of the nucleic acid) in
the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0167] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous
and nonaqueous methods are described in that reference and either
can be used. Specific hybridization conditions referred to herein
are as follows: 1) low stringency hybridization conditions in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at
50.degree. C. (the temperature of the washes can be increased to
55.degree. C. for low stringency conditions); 2) medium stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C.; 3) high stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and preferably 4) very
high stringency hybridization conditions are 0.5M sodium phosphate,
7% SDS at 65.degree. C., followed by one or more washes at
0.2.times.SSC, 1% SDS at 65.degree. C. Very high stringency
conditions (4) are the preferred conditions and the ones that
should be used unless otherwise specified.
[0168] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under a stringency condition described
herein to the sequence of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10, 12, 13,
15, 16 or 18 corresponds to a naturally-occurring nucleic acid
molecule.
[0169] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature. For example a naturally occurring
nucleic acid molecule can encode a natural protein.
[0170] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include at least an open
reading frame encoding a snake venom protease protein. The gene can
optionally further include non-coding sequences, e.g., regulatory
sequences and introns.
[0171] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. "Substantially free" means
that a preparation of a snake venom protease protein is at least
10% pure. In a preferred embodiment, the preparation of snake venom
protease protein has less than about 30%, 20%, 10% and more
preferably 5% (by dry weight), of non-snake venom protease protein
(also referred to herein as a "contaminating protein"), or of
chemical precursors or non-snake venom protease chemicals. When the
snake venom protease protein or biologically active portion thereof
is recombinantly produced, it is also preferably substantially free
of culture medium, i.e., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation. The
invention includes isolated or purified preparations of at least
0.01, 0.1, 1.0, and 10 milligrams in dry weight.
[0172] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a snake venom protease
without abolishing or substantially altering a snake venom protease
activity. Preferably the alteration does not substantially alter
the snake venom protease activity, e.g., the activity is at least
20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid
residue is a residue that, when altered from the wild-type sequence
of a snake venom protease, results in abolishing a snake venom
protease activity such that less than 20% of the wild-type activity
is present. For example, conserved amino acid residues in between
the snake venom proteases, e.g., the snake venom proteases shown in
FIG. 24 are predicted to be particularly unamenable to
alteration.
[0173] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a snake venom protease
protein is preferably replaced with another amino acid residue from
the same side chain family. Alternatively, in another embodiment,
mutations can be introduced randomly along all or part of a snake
venom protease coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be screened for snake venom protease
biological activity to identify mutants that retain activity.
Following mutagenesis of SEQ ID Nos: 1, 3, 4, 6, 7, 9, 10, 12, 13,
15, 16 or 18, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined.
[0174] Amino acid residues can be generally sub-classified into
major subclasses as follows:
[0175] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having an acidic side chain
include glutamic acid and aspartic acid.
[0176] Basic: The residue has a positive charge due to association
with H ion at physiological pH or within one or two pH units
thereof (e.g., histidine) and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having a basic side chain
include arginine, lysine and histidine.
[0177] Charged: The residues are charged at physiological pH and,
therefore, include amino acids having acidic or basic side chains
(i.e., glutamic acid, aspartic acid, arginine, lysine and
histidine).
[0178] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium. Amino acids having
a hydrophobic side chain include tyrosine, valine, isoleucine,
leucine, methionine, phenylalanine and tryptophan.
[0179] Neutral/polar: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium. Amino acids having a neutral/polar side chain
include asparagine, glutamine, cysteine, histidine, serine and
threonine.
[0180] This description also characterises certain amino acids as
"small" since their side chains are not sufficiently large, even if
polar groups are lacking, to confer hydrophobicity. With the
exception of proline, "small" amino acids are those with four
carbons or less when at least one polar group is on the side chain
and three carbons or less when not. Amino acids having a small side
chain include glycine, serine, alanine and threonine. The
gene-encoded secondary amino acid proline is a special case due to
its known effects on the secondary conformation of peptide chains.
The structure of proline differs from all the other
naturally-occurring amino acids in that its side chain is bonded to
the nitrogen of the .alpha.-amino group, as well as the
.alpha.-carbon. Several amino acid similarity matrices (e.g.,
PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff
et al. (1978) A model of evolutionary change in proteins. Matrices
for determining distance relationships In M. O. Dayhoff, (ed.),
Atlas of protein sequence and structure, Vol. 5, pp. 345-358,
National Biomedical Research Foundation, Washington DC; and by
Gonnet et al., 1992, Science 256(5062): 144301445), however,
include proline in the same group as glycine, serine, alanine and
threonine. Accordingly, for the purposes of the present invention,
proline is classified as a "small" amino acid.
[0181] The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and, therefore,
amino acids specifically contemplated by the invention have been
classified as one or the other. Most amino acids not specifically
named can be classified on the basis of known behaviour.
[0182] Amino acid residues can be further sub-classified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxyl carbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always nonaromatic.
[0183] For the naturally occurring protein amino acids,
sub-classification according to the foregoing scheme is presented
in the following Table.
3 Amino acid sub-classification Sub-classes Amino acids Acidic
Aspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine;
Cyclic: Histidine Charged Aspartic acid, Glutamic acid, Arginine,
Lysine, Histidine Small Glycine, Serine, Alanine, Threonine
Polar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,
Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,
Valine, Isoleucine, Leucine, Methionine, Phenylalanine,
Tryptophan
[0184] The gene-encoded secondary amino acid proline is a special
case due to its known effects on the secondary conformation of
peptide chains, and is not, therefore, included in a group.
[0185] The "modified" amino acids that may be included in the SVPs
are gene-encoded amino acids which have been processed after
translation of the gene, e.g., by the addition of methyl groups or
derivatization through covalent linkage to other substituents or
oxidation or reduction or other covalent modification. The
classification into which the resulting modified amino acid falls
will be determined by the characteristics of the modified form. For
example, if lysine were modified by acylating the .epsilon.-amino
group, the modified form would not be classed as basic but as
polar/large.
[0186] Certain commonly encountered amino acids, which are not
encoded by the genetic code, include, for example, .beta.-alanine
(.beta.-Ala), or other omega-amino acids, such as 3-aminopropionic,
2,3-diaminopropionic (2,3-diaP), 4-aminobutyric and so forth,
.alpha.-aminoisobutyric acid (Aib), sarcosine (Sar), omithine
(Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine
(t-BuG), N-methylisoleucine (N-MeIle),, phenylglycine (Phg), and
cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine
(2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);
.beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO); and
homoarginirie (Har). These also fall conveniently into particular
categories.
[0187] Based on the above definitions, Sar, beta-Ala and Aib are
small; t-BuA, t-BuG, N-MeIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic
are hydrophobic; 2,3-diaP, Orn and Har are basic; Cit, Acetyl Lys
and MSO are neutral/polar/large. The various omega-amino acids are
classified according to size as small (.beta.-Ala and
3-aminopropionic) or as large and hydrophobic (all others).
[0188] Other amino acid substitutions for those encoded in the gene
can also be included in SLEs within the scope of the invention and
can be classified within this general scheme according to their
structure.
[0189] In all of the SVPs of the invention, one or more amide
linkages (--CO--NH--) may optionally be replaced with another
linkage which is an isostere such as --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2CH.sub.2, --CH.dbd.CH-- (cis and trans), --COCH.sub.2--,
--CH(OH)CH.sub.2-- and --CH.sub.2SO--. This replacement can be made
by methods known in the art. The following references describe
preparation of peptide analogues which include these
alternative-linking moieties: Spatola, A. F., Vega Data (March
1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general
review); Spatola, A. F., in "Chemistry and Biochemistry of Amino
Acids Peptides and Proteins", B. Weinstein, eds., Marcel Dekker,
New York, p. 267 (1983) (general review); Morley, J. S., Trends
Pharm Sci (1980) pp. 463-468 (general review); Hudson, D., et al.,
Int J Pept Prot Res (1979) 14:177-185 (--CH.sub.2NH--,
--CH.sub.2CH.sub.2--); Spatola, A. F., et al., Life Sci (1986)
38:1243-1249 (--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans
I (1982) 307-314 (--CH--CH--, cis and trans); Almiquist, R. G., et
al., J Med Chem (1980) 23:1392-1398 (--COCH.sub.2--);
Jennings-White, C., et al., Tetrahedron Lett (1982) 23:2533
(--COCH.sub.2--); Szelke, M., et al., European Application EP 45665
(1982) CA:97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay, M. W., et
al., Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH.sub.2--); and
Hruby, V. J., Life Sci (1982) 31: 189-199 (--CH.sub.2--S--).
[0190] As used herein, a "biologically active portion" of a snake
venom protease protein includes a fragment of a snake venom
protease protein which participates in an interaction, e.g., an
intramolecular or an inter-molecular interaction. An
inter-molecular interaction can be a specific binding interaction
or an enzymatic interaction (e.g., the interaction can be transient
and a covalent bond is formed or broken). An inter-molecular
interaction can be between a snake venom protease molecule and a
non-snake venom protease molecule, e.g. prothrombin, or between a
first snake venom protease molecule, e.g., a light chain of a snake
venom protease and a second snake venom protease molecule (e.g., a
dimerization interaction). Biologically active portions of a snake
venom protease protein include peptides comprising amino acid
sequences sufficiently homologous to or derived from the amino acid
sequence of the snake venom protease protein, e.g., the amino acid
sequences shown in SEQ ID NOs:2, 5, 8, 11, 14 or 17, which include
less amino acids than the full length snake venom protease
proteins, and exhibit at least one activity of a snake venom
protease protein. Typically, biologically active portions comprise
a domain or motif with at least one activity of the snake venom
protease protein, e.g., the ability to process prothrombin to
thrombin, e.g., in the absence of calcium and/or phospholipid. A
biologically active portion of a snake venom protease protein can
be a polypeptide which is, for example, 10, 25, 50, 100, 200 or
more amino acids in length. Preferably, said fragment is a
"biologically-active portion" having no less than 1%, preferably no
less than 10%, more preferably no less than 25% and even more
preferably no less than 50% of the prothrombin processing activity
of the snake venom proteases described herein
[0191] The invention contemplates a "fragment" of a snake venom
protease of the invention. The term "fragment" includes within its
scope heavy and light chain fragments of a snake venom protease. In
one embodiment, the fragment is a peptide comprising an amino acid
sequence as shown below (residue numbers as shown in FIG. 27):
4 KREASLPDFVQS (residues 181-192) [SEQ ID NO: 19] LKKSDNPSPDIR
(residues 198-209) [SEQ ID NO: 20] SVXVGEIXXSR (residues 260-270)
[SEQ ID NO: 21] MAPQLLLCLILTFLWSLPEAESNVFLKSK (residues 1-29) [SEQ
ID NO: 22] ANRFLQRTKR (residues 31-40) [SEQ ID NO: 23]
KREASLPDFVQSXXAXXLKKSDNPSPDIIR (residues 181-209) [SEQ ID NO: 24]
MAPQLLLCLILTFLWSLPEAESNVFLKSKXANRFLQRTKR (residues 1-40) [SEQ ID
NO: 25]
[0192] X may be any amino acid.
[0193] It will be appreciated that peptide sub-fragments of the
above peptide fragments are also contemplated, for example peptides
as set forth by SEQ ID NOS: 19 and 20 are respective sub-fragments
of the peptide set forth by SEQ ID NO: 24. Other fragments and
sub-fragments may be selected by a person skilled in the art. In
still another embodiment, a "fragment" is a small peptide, for
example of at least 6, preferably at least 10 and more preferably
at least 20 amino acids in length. Larger fragments comprising more
than one peptide are also contemplated, and may be obtained through
the application of standard recombinant nucleic acid techniques or
synthesized using conventional liquid or solid phase synthesis
techniques. Alternatively, peptides can be produced by digestion of
a polypeptide of the invention with proteinases such as endoLys-C,
endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested
fragments can be purified by, for example, high performance liquid
chromatographic (HPLC) techniques.
[0194] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0195] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, 60%, and even more preferably at
least 70%, 80%, 90%, 100% of the length of the reference sequence.
The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology").
[0196] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0197] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used unless otherwise
specified) are a Blossum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0198] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller ((1989) CABIOS, 4:11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0199] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 53010 nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to 53010 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0200] Particularly preferred snake venom protease polypeptides of
the present invention have an amino acid sequence substantially
identical to the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14
or 17. In the context of an amino acid sequence, the term
"substantially identical" is used herein to refer to a first amino
acid that contains a sufficient or minimum number of amino acid
residues that are i) identical to, or ii) conservative
substitutions of aligned amino acid residues in a second amino acid
sequence such that the first and second amino acid sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NOs:2, 5, 8, 11, 14 or 17 are termed
substantially identical.
[0201] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic acid sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NOs:1, 3, 4, 6, 7, 9, 10, 12, 13, 15,
16, or 18 are termed substantially identical.
[0202] "Subject," as used herein, refers to human and non-human
animals. The term "non-human animals" of the invention includes all
vertebrates, e.g., mammals, such as non-human primates
(particularly higher primates), sheep, dog, rodent (e.g., mouse or
rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals,
such as chickens, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model.
[0203] A "purified preparation of cells", as used herein, refers to
an in vitro preparation of cells. In the case cells from
multicellular organisms (e.g., plants and animals), a purified
preparation of cells is a subset of cells obtained from the
organism, not the entire intact organism. In the case of
unicellular microorganisms (e.g., cultured cells and microbial
cells), it consists of a preparation of at least 10% and more
preferably 50% of the subject cells.
[0204] Variants may fall within the scope of the term "homologs" of
the snake venom protease proteins of the invention.
[0205] As generally used herein, a "homolog" shares a definable
nucleotide or amino acid sequence relationship with a nucleic acid
or amino acid sequence of the invention as the case may be. The
snake venom protease proteins of the invention derived from
different snakes are homologs of each other.
[0206] Included within the scope of homologs are "orthologs", which
are snake venom protease proteins and their encoding nucleic acids,
isolated from organisms other than Pseudonaja textilis, Oxyuranus
scutellatus, Notechis scutatus, Tropidechis carinatus and
Pseudechis porphyriacus. Also, a snake venom protease protein from
one of the above species is an ortholog of any of the other
mentioned species. For example, a snake venom protease protein from
P. textilis is an ortholog of a snake venom protease protein from
O. scutellatus.
[0207] Other derivatives contemplated by the invention include, but
are not limited to, modification to side chains, incorporation of
unnatural amino acids and/or their derivatives during peptide,
polypeptide or protein synthesis and the use of crosslinkers and
other methods which impose conformational constraints on the
polypeptides, fragments and variants of the invention. Examples of
side chain modifications contemplated by the present invention
include modifications of amino groups such as by acylation with
acetic anhydride; acylation of amino groups with succinic anhydride
and tetrahydrophthalic anhydride; amidination with
methylacetimidate; carbamoylation of amino groups with cyanate;
pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4; reductive alkylation by reaction with an
aldehyde followed by reduction with NaBH.sub.4; and
trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene
sulphonic acid (TNBS).
[0208] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivitization, by way of example, to a corresponding amide.
[0209] The guanidine group of arginine residues may be modified by
formation of heterocyclic condensation products with reagents such
as 2,3-butanedione, phenylglyoxal and glyoxal.
[0210] Sulphydryl groups may be modified by methods such as
performic acid oxidation to cysteic acid; formation of mercurial
derivatives using 4-chloromercuriphenylsulphonic acid,
4-chloromercuribenzoate; 2-chloromercuri-4-nitrophenol,
phenylmercury chloride, and other mercurials; formation of a mixed
disulphides with other thiol compounds; reaction with maleimide,
maleic anhydride or other substituted maleimide; carboxymethylation
with iodoacetic acid or iodoacetamide; and carbamoylation with
cyanate at alkaline pH.
[0211] Tryptophan residues may be modified, for example, by
alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide
or sulphonyl halides or by oxidation with N-bromosuccinimide.
[0212] Tyrosine residues may be modified by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0213] The imidazole ring of a histidine residue may be modified by
N-carbethoxylation with diethylpyrocarbonate or by alkylation with
iodoacetic acid derivatives.
[0214] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include but are not limited
to, use of 4-amino butyric acid, 6-aminohexanoic acid,
4-amino-3-hydroxy-5-phenylpentanoic acid,
4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine,
norleucine, norvaline, phenylglycine, omithine, sarcosine,
2-thienyl alanine and/or D-isomers of amino acids.
[0215] Isolated prothrombin activating proteins of the invention
(inclusive of fragments, variants, derivatives and homologs) may be
prepared by any suitable procedure known to those of skill in the
art.
[0216] Various aspects of the invention are described in further
detail below.
[0217] Isolated nucleic acid molecules
[0218] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a snake venom protease
polypeptide described herein, e.g., a full-length snake venom
protease protein or a fragment thereof, e.g., a biologically active
portion of snake venom protease protein. Also included is a nucleic
acid fragment suitable for use as a hybridization probe, which can
be used, e.g., to identify a nucleic acid molecule encoding a
polypeptide of the invention, snake venom protease mRNA, and
fragments suitable for use as primers, e.g., PCR primers for the
amplification or mutation of nucleic acid molecules.
[0219] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NOs:1,
3, 4, 6, 7, 9, 10, 12, 13, 15, 16, or 18, or a portion of any of
these nucleotide sequences. In one embodiment, the nucleic acid
molecule includes sequences encoding the snake venom protease
protein (i.e., "the coding region" of SEQ ID NO:1, 4, 7, 10, 13 or
16, as shown in SEQ ID NO:3, 6, 9, 12, 15 or 18, respectively), as
well as 5' untranslated sequences. Alternatively, the nucleic acid
molecule can include only the coding region of SEQ ID NO:1, 4, 7,
10, 13 or 16 (e.g., SEQ ID NO:3, 6, 9, 12, 15 or 18, respectively)
and, e.g., no flanking sequences which normally accompany the
subject sequence. In another embodiment, the nucleic acid molecule
encodes a sequence corresponding to a fragment of the protein. For
example, the nucleic acid molecule encodes one or more of a snake
venom protease propeptide, light chain, activation peptide and
heavy chain. In another embodiment, the nucleic acid molecule can
encode on or more of the domains or regions described herein.
[0220] In another embodiment, an isolated nucleic acid molecule of
the invention includes a nucleic acid molecule which is a
complement, e.g., a full complement, of the nucleotide sequence
shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, or 18, or
a portion of any of these nucleotide sequences, e.g., any portion
encoding a domain or region described herein. In other embodiments,
the nucleic acid molecule of the invention is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:1, 3,
4, 6, 7, 9, 10, 12, 13, 15, 16, or 18, such that it can hybridize
(e.g., under a stringency condition described herein) to the
nucleotide sequence shown in SEQ ID NO:1, 3, 4,6 ,7, 9, 10, 12, 13,
15, 16, or 18, thereby forming a stable duplex.
[0221] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12,
13, 15, 16, or 18, or a portion, preferably of the same length, of
any of these nucleotide sequences.
[0222] Snake Venom Protease Nucleic Acid Fragments
[0223] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 7, 9,
10, 12, 13, 15, 16, or 18. For example, such a nucleic acid
molecule can include a fragment which can be used as a probe or
primer or a fragment encoding a portion of a snake venom protease
protein, e.g., an immunogenic or biologically active portion of a
snake venom protease protein, e.g., an immunogenic or biologically
active portion of a snake venom protease protein described herein.
A fragment can comprise those nucleotides of SEQ ID NO:1, 3, 4, 6,
7, 9, 10, 12, 13, 15, 16, or 18, which encodes, e.g., a propeptide,
a light chain, an activator peptide, a heavy chain, a GLA domain,
an EGF-1 domain, an EGF-2 domain, or any other domain or region
described herein, of snake venom protease. The nucleotide sequence
determined from the cloning of the snake venom protease gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning other snake venom protease family
members, or fragments thereof, as well as snake venom protease
homologues, or fragments thereof, from other species.
[0224] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding region and
extends into either (or both) the 5' or 3' noncoding region. Other
embodiments include a fragment which includes a nucleotide sequence
encoding an amino acid fragment described herein. Nucleic acid
fragments can encode a specific domain or site described herein or
fragments thereof, particularly fragments thereof which are at
least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino
acids in length. Fragments also include nucleic acid sequences
corresponding to specific amino acid sequences described above or
fragments thereof. Nucleic acid fragments should not to be
construed as encompassing those fragments that may have been
disclosed prior to the invention.
[0225] A nucleic acid fragment can include a sequence corresponding
to a domain, region, or functional site described herein. A nucleic
acid fragment can also include one or more domain, region, or
functional site described herein. Thus, for example, a snake venom
protease nucleic acid fragment can include a sequence corresponding
to a GLA domain, an EGF domain or a factor Va-like domain.
[0226] Snake venom protease probes and primers are provided.
Typically a probe/primer is an isolated or purified
oligonucleotide. The oligonucleotide typically includes a region of
nucleotide sequence that hybridizes under a stringency condition
described herein to at least about 7, 12 or 15, preferably about 20
or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75
consecutive nucleotides of a sense or antisense sequence of SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 and/or 18, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:1, 3, 4,
6, 7, 9, 10, 12, 13, 15, 16, or 18. Preferably, an oligonucleotide
is less than about 200, 150, 120, or 100 nucleotides in length. In
a preferred embodiment, the snake venom protease probes or primers
hybrize to a region of a snake venom protease encoding nucleic acid
but do not hybridize to a region of human factor Xa and/or
trocarin.
[0227] In one embodiment, the probe or primer is attached to a
solid support, e.g., a solid support described herein.
[0228] One exemplary kit of primers includes a forward primer that
anneals to the coding strand and a reverse primer that anneals to
the non-coding strand. The forward primer can anneal to the start
codon, e.g., the nucleic acid sequence encoding amino acid residue
1 of SEQ ID NO:2, 5, 8, 11, 14 or 17. The reverse primer can anneal
to the ultimate codon, e.g., the codon immediately before the stop
codon, e.g., the codon encoding amino acid residue 581 of SEQ ID
NO:2, 5, 8, 11, 14, or 17. In a preferred embodiment, the annealing
temperatures of the forward and reverse primers differ by no more
than 5, 4, 3, or 2.degree. C.
[0229] In a preferred embodiment the nucleic acid is a probe which
is at least 10, 12, 15, 18, 20 and less than 200, more preferably
less than 100, or less than 50, nucleotides in length. It should be
identical, or differ by 1, or 2, or less than 5 or 10 nucleotides,
from a sequence disclosed herein. If alignment is needed for this
comparison the sequences should be aligned for maximum homology.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences.
[0230] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes: a propeptide, a
light chain, an activator peptide, a heavy chain, or portions
thereof (or domains within such regions).
[0231] In another embodiment a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
selected region of a snake venom protease sequence, e.g., a domain,
region, site or other sequence described herein. The primers should
be at least 5, 10, or 50 base pairs in length and less than 100, or
less than 200, base pairs in length. The primers should be
identical, or differs by one base from a sequence disclosed herein
or from a naturally occurring variant. For example, primers
suitable for amplifying all or a portion of any of the following
regions are provided: a propeptide, a light chain, an activator
peptide, a heavy chain (or domains and sites within those
regions).
[0232] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0233] A nucleic acid fragment encoding a "biologically active
portion of a snake venom protease polypeptide" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO:1, 3,
4, 6, 7, 9, 10, 12, 13, 15, 16, or 18, which encodes a polypeptide
having a snake venom protease biological activity (e.g., the
biological activities of the snake venom protease proteins are
described herein), expressing the encoded portion of the snake
venom protease protein (e.g., by recombinant expression in vitro)
and assessing the activity of the encoded portion of the snake
venom protease protein. A nucleic acid fragment encoding a
biologically active portion of a snake venom protease polypeptide,
may comprise a nucleotide sequence which is greater than 300 or
more nucleotides in length.
[0234] In preferred embodiments, a nucleic acid includes a
nucleotide sequence which is about 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400 or more nucleotides in length and
hybridizes under a stringency condition described herein to a
nucleic acid molecule of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13,
15, 16, or 18.
[0235] Snake Venom Protease Nucleic Acid Variants
[0236] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, 3,
4, 6, 7, 9, 10, 12, 13, 15, 16 or 18. Such differences can be due
to degeneracy of the genetic code (and result in a nucleic acid
which encodes the same snake venom protease proteins as those
encoded by the nucleotide sequence disclosed herein. In another
embodiment, an isolated nucleic acid molecule of the invention has
a nucleotide sequence encoding a protein having an amino acid
sequence which differs, by at least 1, but less than 5, 10, 20, 50,
or 100 amino acid residues that shown in SEQ ID NO:2, 5, 8, 11, 14
or 17. If alignment is needed for this comparison the sequences
should be aligned for maximum homology. The encoded protein can
differ by no more than 5, 4, 3, 2, or 1 amino acid. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0237] Nucleic acids of the invention can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. E.g., the nucleic acid can be one in which at
least one codon, at preferably at least 10%, or 20% of the codons
has been altered such that the sequence is optimized for expression
in E. coli, yeast, human, insect, or CHO cells.
[0238] Nucleic acid variants can be naturally occurring, such as
allelic variants (sarne locus), homologs (different locus), and
orthologs (different organism) or can be non naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product). In one
embodiment, nucleic acid homologs are orthologous nucleic acids
isolated from snakes other than Pseudonaja textilis, Oxyuranus
scutellatus, Notechis scutatus, Tropidechis carinatus and
Pseudechis porphyriacus.
[0239] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18,
e.g., as follows: by at least one but less than 10, 20, 30, or 40
nucleotides; at least one but less than 1%, 5%, 10% or 20% of the
nucleotides in the subject nucleic acid. The nucleic acid can
differ by no more than 5, 4, 3, 2, or 1 nucleotide. If necessary
for this analysis the sequences should be aligned for maximum
homology. "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.
[0240] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is typically at
least about 70-75%, more typically at least about 80-85%, and most
typically at least about 90-95% or more identical to the nucleotide
sequence shown in SEQ ID NO:2, 5, 8, 11, 14 or 17 or a fragment of
this sequence and preferably has a snake venom protease activity.
Such nucleic acid molecules can readily be identified as being able
to hybridize under a stringency condition described herein, to the
nucleotide sequence shown in SEQ ID NO 2, 5, 8, 11, 14, 17, or a
fragments thereof. Nucleic acid molecules corresponding to
orthologs, homologs, and allelic variants of the snake venom
protease cDNAs of the invention can further be isolated by mapping
to the same chromosome or locus as the snake venom protease
gene.
[0241] Preferred variants include those that have a snake venom
protease activity, e.g., an ability to induce clotting in the
absence of one or more of calcium, phospholipid and factor Va.
[0242] Allelic variants of snake venom protease include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the snake
venom protease protein within a population that maintain the
ability to process prothrombin. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:2, 5, 8, 11, 14 or 17, or substitution,
deletion or insertion of non-critical residues in non-critical
regions of the protein. Non-functional allelic variants are
naturally-occurring amino acid sequence variants of the snake venom
protease protein within a population that do not have the ability
to process prothrombin. Non-functional allelic variants will
typically contain a non-conservative substitution, a deletion, or
insertion, or premature truncation of the amino acid sequence of
SEQ ID NO:2, 5, 8, 11, 14, 17, or a substitution, insertion, or
deletion in critical residues or critical regions of the
protein.
[0243] Moreover, nucleic acid molecules encoding other snake venom
protease family members and, thus, which have a nucleotide sequence
which differs from the snake venom protease sequences of SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16 or 18 are intended to be
within the scope of the invention.
[0244] Isolated nucleic acid homologs of the invention may also be
prepared by methods utilizing nucleic acid sequence amplification
techniques.
[0245] In one embodiment, the method includes the steps of:
[0246] (i) obtaining a nucleic acid extract from a host cell or
animal;
[0247] (ii) creating one or more primers which, optionally, are
degenerate wherein each said primer corresponds to a portion of an
isolated nucleic acid of the invention; and
[0248] (iii) using said primers to amplify, via a nucleic acid
amplification technique, one or more amplification products from
said nucleic acid extract.
[0249] Suitably, said one or more primers are designed to be
capable of annealing to one or the other strands of a
double-stranded nucleic acid of the invention under annealing and
primer extension conditions typically used for amplification. In
the case of degenerate primers, sequence differences between the
primer and the isolated nucleic acid sequence are intentionally
introduced to account for possible sequence variation, such as due
to degeneracy in homologous coding sequences.
[0250] Suitable nucleic acid amplification techniques are well
known to the skilled addressee, and include polyrnerase chain
reaction (PCR) and ligase chain reaction (LCR) as for example
described in Chapter 15 of Ausubel et al. supra; strand
displacement amplification (SDA) as for example described in U.S.
Pat. No. 5,422,252; rolling circle replication (RCR) as for example
described in International application WO 92/01813 and
International Application WO 97/19193; nucleic acid sequence-based
amplification (NASBA) as for example described by Sooknanan et al.,
1994, Biotechniques 17 1077; and Q-.beta. replicase amplification
as for example described by Tyagi et al., 1996, Proc. Natl. Acad.
Sci. USA 93 5395, although without limitation thereto.
[0251] A preferred nucleic acid sequence amplification technique is
PCR.
[0252] As used herein, an "amplification product" refers to a
nucleic acid product generated by a nucleic acid amplification
technique as broadly defined herein.
[0253] A nucleic acid homolog may encode a protein homolog.
Accordingly, the above-described methods for isolating a nucleic
acid homolog may be used to isolate a protein homolog.
[0254] Isolated Snake Venom Protease Polypeptides
[0255] In another aspect, the invention features, an isolated snake
venom protease protein, or fragment, e.g., a biologically active
portion, for use as immunogens or antigens to raise or test (or
more generally to bind) anti-snake venom protease antibodies. The
snake venom protease protein can be isolated from cells or tissue
sources using standard protein purification techniques. In one
embodiment, the snake venom protease is isolated from a snake
selected from the group of: Pseudonaja textilis, Oxyuranus
scutellatus, Notechis scutatus, Tropidechis carinatus and
Pseudechis porphyriacus. Preferably, the snake venom protease is
isolated from the venom gland of an Australian snake, e.g., an
Australian snake described herein. Snake venom protease protein or
fragments thereof can be produced by recombinant DNA techniques or
synthesized chemically.
[0256] Polypeptides of the invention include those which arise as a
result of the existence of alternative translational and
post-translational events. The polypeptide can be expressed in
systems, e.g., cultured cells, which result in substantially the
same post-translational modifications present when expressed the
polypeptide is expressed in a native cell, or in systems which
result in the alteration or omission of post-translational
modifications, e.g., glycosylation or cleavage, present when
expressed in a native cell.
[0257] In a preferred embodiment, a snake venom protease
polypeptide has one or more of the following characteristics:
[0258] (i) it has the ability to process prothrombin;
[0259] (ii) it has a molecular weight, e.g., a deduced molecular
weight, preferably ignoring any contribution of post translational
modifications, amino acid composition or other physical
characteristic of a snake venom protease polypeptide, e.g., a
polypeptide of SEQ ID NO:2, 5, 8, 11, 14 or 17;
[0260] (iii) it has an overall sequence similarity of at least 60%,
more preferably at least 70, 80, 90, or 95%, with a snake venom
protease polypeptide, e.g., a polypeptide of SEQ ID NO:2, 5, 8, 11,
14 or 17;
[0261] (iv) it has a substantial sequence identity with one or more
of the domains or regions described herein, e.g., as described
herein.
[0262] In a preferred embodiment, the snake venom protease protein,
or fragment thereof, differs from the corresponding sequence in SEQ
ID NO:2, 5, 8, 11, 14, or 17. In one embodiment, it differs by at
least one but by less than 15, 10 or 5 amino acid residues. In
another, it differs from the corresponding sequence in SEQ ID NO:2,
5, 8, 11, 14 or 17 by at least one residue but less than 20%, 15%,
10% or 5% of the residues in it differ from the corresponding
sequence in SEQ ID NO:2, 5, 8, 11, 14 or 17. (If this comparison
requires alignment the sequences should be aligned for maximum
homology. "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.) The differences are,
preferably, differences or changes at a non-essential residue or a
conservative substitution.
[0263] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such snake venom
protease proteins differ in amino acid sequence from SEQ ID NO:2,
5, 8, 11, 14 or 17, yet retain biological activity.
[0264] In one embodiment, the protein includes an amino acid
sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or more homologous to SEQ ID NO:2, 5, 8, 11, 14 or
17, and has a snake venom protease biological activity.
[0265] In one embodiment, a biologically active portion of a snake
venom protease protein includes one or more of: a GLA domain, an
EGF-1 domain, an EGF-2 domain and a factor Va-like domain.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native snake venom protease protein.
[0266] In a preferred embodiment, the snake venom protease protein
has an amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, 14 or
17. In other embodiments, the snake venom protease protein is
substantially identical to SEQ ID NO:2, 5, 8, 11, 14, or 17, and
retains the functional activity of the protein of SEQ ID NO:2, 5,
8, 11, 14 or 17, as described in detail in the subsections above.
In a preferred embodiment, the snake venom protease protein retains
the ability to process prothrombin in the absence of one or more of
calcium, phospholipids and factor Va, preferably it retains the
ability to process prothrombin in the absence or both calcium and
phospholipid.
[0267] Snake Venom Protease Chimeric or Fusion Proteins
[0268] In another aspect, the invention provides snake venom
protease chimeric or fusion proteins. As used herein, a snake venom
protease "chimeric protein" or "fusion protein" includes a snake
venom protease polypeptide linked to a non-snake venom protease
polypeptide. A "non-snake venom protease polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is different from the snake venom protease protein
and which is derived from the same or a different organism. The
snake venom protease polypeptide of the fusion protein can
correspond to all or a portion e.g., a fragment described herein of
a snake venom protease amino acid sequence. In a preferred
embodiment, a snake venom protease fusion protein includes at least
one (or two) biologically active portion of a snake venom protease
protein. The non-snake venom protease polypeptide can be fused to
the N-terminus or C-terminus of the snake venom protease
polypeptide. In one embodiment, the "non-snake venom protease
polypeptide" is a pro-peptide from a prothrombotic activating
protein other than a snake venom protease, e.g., it is a propeptide
from mammalian factor Xa, e.g., human factor Xa. In another
embodiment, the "non-snake venom protease polypeptide" can include
an activator peptide from a prothrombotic activating protein other
than a snake venom protease, e.g., an activator peptide from
mammalian factor Xa, e.g., human factor Xa. In yet another
embodiment, the chimeric or fusion polypeptide can include a
propeptide and an activator peptide from a "non-snake venom
protease polypeptide", e.g., from a mammalian factor Xa
polypeptide, e.g., a human factor Xa polypeptide.
[0269] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-snake venom protease fusion protein in which the snake venom
protease sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant snake venom protease. Alternatively, the fusion protein
can be a snake venom protease protein containing a heterologous
signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host cells), expression and/or secretion of snake venom
protease can be increased through use of a heterologous signal
sequence.
[0270] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0271] The snake venom protease fusion proteins of the invention
can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The snake venom protease fusion
proteins can be used to affect the bioavailability of a snake venom
protease substrate.
[0272] Moreover, the snake venom protease-fusion proteins of the
invention can be used as immunogens to produce anti-snake venom
protease antibodies in a subject, to purify snake venom protease
ligands and in screening assays to identify molecules which inhibit
the interaction of snake venom protease with a snake venom protease
substrate.
[0273] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A snake venom
protease-encoding nucleic acid can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to
the snake venom protease protein.
[0274] Variants of Snake Venom Protease Proteins
[0275] In another aspect, the invention also features a variant of
a snake venom protease polypeptide, e.g., which functions as an
agonist (mimetics) or as an antagonist. Variants of the snake venom
protease proteins can be generated by mutagenesis, e.g., discrete
point mutation, the insertion or deletion of sequences or the
truncation of a snake venom protease protein. An agonist of the
snake venom protease proteins can retain substantially the same, or
a subset, of the biological activities of the naturally occurring
form of a snake venom protease protein. An antagonist of a snake
venom protease protein can inhibit one or more of the activities of
the naturally occurring form of the snake venom protease protein
by, for example, competitively modulating a snake venom
protease-mediated activity of a snake venom protease protein. Thus,
specific biological effects can be elicited by treatment with a
variant of limited function.
[0276] Variants of a snake venom protease protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation
mutants, of a snake venom protease protein for agonist or
antagonist activity.
[0277] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a snake venom protease protein coding
sequence can be used to generate a variegated population of
fragments for screening and subsequent selection of variants of a
snake venom protease protein. Variants in which a cysteine residues
is added or deleted, in which a calcium binding residue, e.g., a
carboxyglutamic acid residue or asparganine, is added or deleted or
in which a residue which is glycosylated is added or deleted are
particularly preferred.
[0278] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property are
known in the art. Such methods are adaptable for rapid screening of
the gene libraries generated by combinatorial mutagenesis of snake
venom protease proteins. Recursive ensemble mutagenesis (REM), a
new technique which enhances the frequency of functional mutants in
the libraries, can be used in combination with the screening assays
to identify snake venom protease variants (Arkin and Yourvan (1992)
Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993)
Protein Engineering 6:327-331).
[0279] In another aspect, the invention features a method of making
a snake venom protease polypeptide, e.g., a peptide having a
non-wild type activity, e.g., an antagonist, agonist, or super
agonist of a naturally occurring snake venom protease polypeptide,
e.g., a naturally occurring snake venom protease polypeptide. The
method includes: altering the sequence of a snake venom protease
polypeptide, e.g., altering the sequence, e.g., by substitution or
deletion of one or more residues of a non-conserved region, a
domain or residue disclosed herein, and testing the altered
polypeptide for the desired activity.
[0280] In another aspect, the invention features a method of making
a fragment or analog of a snake venom protease polypeptide having a
biological activity of a naturally occurring snake venom protease
polypeptide. The method includes: altering the sequence, e.g., by
substitution or deletion of one or more residues, of a snake venom
protease polypeptide, e.g., altering the sequence of a
non-conserved region, or a domain or residue described herein, and
testing the altered polypeptide for the desired activity.
[0281] Anti-Snake Venom Protease Antibodies
[0282] In another aspect, the invention provides an anti-snake
venom protease antibody, or a fragment thereof (e.g., an
antigen-binding fragment thereof). The term "antibody" as used
herein refers to an immunoglobulin molecule or immunologically
active portion thereof, i.e., an antigen-binding portion. As used
herein, the term "antibody" refers to a protein comprising at least
one, and preferably two, heavy (H) chain variable regions
(abbreviated herein as VH), and at least one and preferably two
light (L) chain variable regions (abbreviated herein as VL). The VH
and VL regions can be further subdivided into regions of
hypervariability, termed "complementarity determining regions"
("CDR"), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the framework region and
CDR's has been precisely defined (see, Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917,
which are incorporated herein by reference). Each VH and VL is
composed of three CDR's and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0283] The anti-snake venom protease antibody can further include a
heavy and light chain constant region, to thereby form a heavy and
light immunoglobulin chain, respectively. In one embodiment, the
antibody is a tetramer of two heavy immunoglobulin chains and two
light immunoglobulin chains, wherein the heavy and light
immunoglobulin chains are inter-connected by, e.g., disulfide
bonds. The heavy chain constant region is comprised of three
domains, CH1, CH2 and CH3. The light chain constant region is
comprised of one domain, CL. The variable region of the heavy and
light chains contains a binding domain that interacts with an
antigen. The constant regions of the antibodies typically mediate
the binding of the antibody to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system.
[0284] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. The recognized human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 KDa or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-- terminus. Full-length
immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids),
are similarly encoded by a variable region gene (about 116 amino
acids) and one of the other aforementioned constant region genes,
e.g., gamma (encoding about 330 amino acids).
[0285] The term "antigen-binding fragment" of an antibody (or
simply "antibody portion," or "fragment"), as used herein, refers
to one or more fragments of a full-length antibody that retain the
ability to specifically bind to the antigen, e.g., snake venom
protease polypeptide or fragment thereof. Examples of
antigen-binding fragments of the anti-snake venom protease antibody
include, but are not limited to: (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
encompassed within the term "antigen-binding fragment" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those with skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0286] The anti-snake venom protease antibody can be a polyclonal
or a monoclonal antibody. In other embodiments, the antibody can be
recombinantly produced, e.g., produced by phage display or by
combinatorial methods.
[0287] Phage display and combinatorial methods for generating
anti-snake venom protease antibodies are known in the art (as
described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et
al. International Publication No. WO 92/18619; Dower et al.
International Publication No. WO 91/17271; Winter et al.
International Publication WO 92/20791; Markland et al.
International Publication No. WO 92/15679; Breitling et al.
International Publication WO 93/01288; McCafferty et al.
International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Ladner et al.
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 2:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the
contents of all of which are incorporated by reference herein).
[0288] In preferred embodiments an antibody can be made by
immunizing with purified snake venom protease antigen, or a
fragment thereof, e.g., a fragment described herein, tissue, e.g.,
crude tissue preparations, whole cells, preferably living cells,
lysed cells, or cell fractions.
[0289] A full-length snake venom protease protein or, antigenic
peptide fragment of a snake venom protease can be used as an
immunogen or can be used to identify anti-snake venom protease
antibodies made with other immunogens, e.g., cells, membrane
preparations, and the like. The antigenic peptide of snake venom
protease should include at least 8 amino acid residues of the amino
acid sequence shown in SEQ ID NO:2, 5, 8, 11, 14 or 17 and
encompasses an epitope of a snake venom protease. Preferably, the
antigenic peptide includes at least 10 amino acid residues, more
preferably at least 15 amino acid residues, even more preferably at
least 20 amino acid residues, and most preferably at least 30 amino
acid residues. In preferred embodiments, the anti-snake venom
protease antibody binds to a region, domain or site of a snake
venom protease described herein. Antibodies reactive with, or
specific for, any of these regions, or other regions or domains
described herein are provided.
[0290] Antibodies which bind only native snake venom protease
protein, only denatured or otherwise non-native snake venom
protease protein, or which bind both, are with in the invention.
Antibodies with linear or conformational epitopes are within the
invention. Conformational epitopes can sometimes be identified by
identifying antibodies which bind to native but not denatured snake
venom protease protein.
[0291] Preferred epitopes encompassed by the antigenic peptide are
regions of snake venom proteases which are located on the light or
heavy chain, hydrophilic regions, as well as regions with high
antigenicity.
[0292] In preferred embodiments, antibodies can bind one or more of
purified antigen, tissue, e.g., tissue sections, whole cells,
preferably living cells, lysed cells, or cell fractions.
[0293] The anti-snake venom protease antibody can be a single chain
antibody. A single-chain antibody (scFV) may be engineered (see,
for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80;
and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain
antibody can be dimerized or multimerized to generate multivalent
antibodies having specificities for different epitopes of the same
target snake venom protease protein.
[0294] The antibody can be coupled to a compound, e.g., a label
such as a radioactive nucleus, or imaging agent, e.g. a
radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR
contrast agent. Labels which produce detectable radioactive
emissions or fluorescence are preferred.
[0295] An anti-snake venom protease antibody (e.g., monoclonal
antibody) can be used to isolate a snake venom protease by standard
techniques, such as affinity chromatography or immunoprecipitation.
Moreover, an anti-snake venom protease antibody can be used to
detect snake venom protease protein (e.g., in a cellular lysate or
cell supernatant) in order to evaluate the abundance and pattern of
expression of the protein. Anti-snake venom protease antibodies can
be used diagnostically to monitor snake venom protease levels in
tissue as part of a clinical testing procedure. Detection can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance (i.e., antibody labeling). Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H. The
label may be selected from a group including a chromogen, a
catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a
lanthanide ion such as Europium (Eu.sup.34), a radioisotope and a
direct visual label. In the case of a direct visual label, use may
be made of a colloidal metallic or non-metallic particle, a dye
particle, an enzyme or a substrate, an organic polymer, a latex
particle, a liposome, or other vesicle containing a signal
producing substance and the like.
[0296] A large number of enzymes useful as labels is disclosed in
U.S. patent Specifications U.S. Pat. No. 4,366,241, U.S. Pat. No.
4,843,000, and U.S. Pat. No. 4,849,338, all of which are herein
incorporated by reference. Enzyme labels useful in the present
invention include alkaline phosphatase, horseradish peroxidase,
luciferase, b-galactosidase, glucose oxidase, lysozyme, malate
dehydrogenase and the like. The enzyme label may be used alone or
in combination with a second enzyme in solution.
[0297] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0298] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0299] A vector can include a snake venom protease nucleic acid in
a form suitable for expression of the nucleic acid in a host cell.
Preferably the recombinant expression vector includes one or more
regulatory sequences operatively linked to the nucleic acid
sequence to be expressed. The term "regulatory sequence" includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
snake venom protease proteins, fusion proteins, and the like).
[0300] The recombinant expression vectors of the invention can be
designed for expression of snake venom protease proteins in
prokaryotic or eukaryotic cells. For example, polypeptides of the
invention can be expressed in E. coli, insect cells (e.g., using
baculovirus expression vectors), yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, (1990) Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. Alternatively, the recombinant expression vector
can be transcribed and translated in vitro, for example using T7
promoter regulatory sequences and T7 polymerase.
[0301] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0302] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S.,
(1990) Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0303] The snake venom protease expression vector can be a yeast
expression vector, a vector for expression in insect cells, e.g., a
baculovirus expression vector or a vector suitable for expression
in mammalian cells.
[0304] When used in mammalian cells, the expression vector's
control functions can be provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0305] In another embodiment, the promoter is an inducible
promoter, e.g., a promoter regulated by a steroid hormone, by a
polypeptide hormone (e.g., by means of a signal transduction
pathway), or by a heterologous polypeptide (e.g., the
tetracycline-inducible systems, "Tet-On" and "Tet-Off"; see, e.g.,
Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).
[0306] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0307] In some embodiments, when used in a mammalian cell, the
expression vector can provide for expression of the snake venom
protease light chain and heavy chain and expression of a propeptide
domain and/or activation peptide from a non-snake venom protease
polypeptide, e.g., a non-snake venom protease prothrombin
activating protein, e.g., a propeptide and/or activation peptide
from a mammalian factor X, e.g., human factor X.
[0308] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus.
[0309] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a snake
venom protease nucleic acid molecule within a recombinant
expression vector or a snake venom protease nucleic acid molecule
containing sequences which allow it to homologously recombine into
a specific site of the host cell's genome. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. Such
terms refer not only to the particular subject cell but to the
progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0310] A host cell can be any prokaryotic or eukaryotic cell. For
example, a snake venom protease protein can be expressed in
bacterial cells (such as E. coli), insect cells, yeast or mammalian
cells (such as Chinese hamster ovary cells (CHO) or COS cells
(African green monkey kidney cells CV-1 origin SV40 cells; Gluzman
(1981) CellI23:175-182)). Other suitable host cells are known to
those skilled in the art.
[0311] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0312] A host cell of the invention can be used to produce (i.e.,
express) a snake venom protease protein. Accordingly, the invention
further provides methods for producing a snake venom protease
protein, e.g., a snake venom protease protein described herein,
using the host cells of the invention. In one embodiment, the
method includes culturing the host cell of the invention (into
which a recombinant expression vector encoding a snake venom
protease protein has been introduced) in a suitable medium such
that a snake venom protease protein is produced. In another
embodiment, the method further includes isolating a snake venom
protease protein from the medium or the host cell.
[0313] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject snake venom protease polypeptide.
[0314] Informatics
[0315] The sequence of a snake venom protease is provided in a
variety of media to facilitate use thereof. A recorded sequence, in
contrast to a protein or nucleic acid, can be provided as a
manufacture. Such a manufacture can provide a nucleotide or amino
acid sequence, e.g., an open reading frame, in a form which allows
examination, e.g., by sequence analysis programs or by direct
inspection, of the manufacture using means not directly applicable
to examining the nucleotide or amino acid sequences, or a subset
thereof, as they exists in nature or in purified form. The sequence
information can include, but is not limited to, SVP full-length
nucleotide and/or amino acid sequences, partial nucleotide and/or
amino acid sequences, polymorphic sequences including single
nucleotide polymorphisms (SNPs), epitope or domain sequence, and
the like. In a preferred embodiment, the manufacture is a
machine-readable medium, e.g., a magnetic, optical, chemical or
mechanical information storage device.
[0316] As used herein, "machine-readable media" refers to any
medium that can be read and accessed directly by a machine, e.g., a
digital computer or analogue computer. Non-limiting examples of a
computer include a desktop PC, laptop, mainframe, server (e.g., a
web server, network server, or server farm), handheld digital
assistant, pager, mobile telephone, and the like. The computer can
be stand-alone or connected to a communications network, e.g., a
local area network (such as a VPN or intranet), a wide area network
(e.g., an Extranet or the Internet), or a telephone network (e.g.,
a wireless, DSL, or ISDN network). Machine-readable media include,
but are not limited to: magnetic storage media, such as floppy
discs, hard disc storage medium, and magnetic tape; optical storage
media such as CD-ROM; electrical storage media such as RAM, ROM,
EPROM, EEPROM, flash memory, and the like; and hybrids of these
categories such as magnetic/optical storage media.
[0317] A variety of data storage structures are available to a
skilled artisan for creating a machine-readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0318] In a preferred embodiment, the sequence information is
stored in a relational database (such as Sybase or Oracle). The
database can have a first table for storing sequence (nucleic acid
and/or amino acid sequence) information. The sequence information
can be stored in one field (e.g., a first column) of a table row
and an identifier for the sequence can be store in another field
(e.g., a second column) of the table row. The database can have a
second table, e.g., storing annotations. The second table can have
a field for the sequence identifier, a field for a descriptor or
annotation text (e.g., the descriptor can refer to a functionality
of the sequence, a field for the initial position in the sequence
to which the annotation refers, and a field for the ultimate
position in the sequence to which the annotation refers.
Non-limiting examples for annotations to amino acid sequence
include polypeptide domains, e.g., a domain described herein;
active sites and other functional amino acids; and modification
sites.
[0319] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. A search is used to identify fragments or regions of the
sequences of the invention which match a particular target sequence
or target motif. The search can be a BLAST search or other routine
sequence comparison, e.g., a search described herein.
[0320] Thus, in one aspect, the invention features a method of
analyzing an SVP sequence, e.g., analyzing structure, function, or
relatedness to one or more other nucleic acid or amino acid
sequences. The method includes: providing a SVP nucleic acid or
amino acid sequence; comparing the SVP sequence with a second
sequence, e.g., one or more preferably a plurality of sequences
from a collection of sequences, e.g., a nucleic acid or protein
sequence database to thereby analyze SVP. The method can be
performed in a machine, e.g., a computer, or manually by a skilled
artisan.
[0321] The method can include evaluating the sequence identity
between a SVP sequence and a second sequence, e.g., database
sequence. The method can be performed by accessing the database at
a second site, e.g., over the Internet.
[0322] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. Typical sequence
lengths of a target sequence are from about 10 to 100 amino acids
or from about 30 to 300 nucleotide residues. However, it is well
recognized that commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, may
be of shorter length.
[0323] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).
[0324] Thus, the invention features a method of making a computer
readable record of a sequence of a SVP sequence which includes
recording the sequence on a computer readable matrix. In a
preferred embodiment the record includes one or more of the
following: identification of an ORF; identification of a domain,
region, or site; identification of the start of transcription;
identification of the transcription terminator; the full length
amino acid sequence of the protein, or a mature form thereof; the
5' end of the translated region.
[0325] In another aspect, the invention features, a method of
analyzing a sequence. The method includes: providing a SVP
sequence, or record, in machine-readable form; comparing a second
sequence to the SVP sequence, e.g., analyzing the SVP sequence for
the presence or absence of a particular motif or domain; thereby
analyzing a sequence. Comparison can include comparing to sequences
for sequence identity or determining if one sequence is included
within the other, e.g., determining if the SVP sequence includes a
sequence being compared. In a preferred embodiment the SVP or
second sequence is stored on a first computer, e.g., at a first
site and the comparison is performed, read, or recorded on a second
computer, e.g., at a second site. E.g., the SVP or second sequence
can be stored in a public or proprietary database in one computer,
and the results of the comparison performed, read, or recorded on a
second computer. In a preferred embodiment the record includes one
or more of the following: identification of an ORF; identification
of a domain, region, or site; identification of the start of
transcription; identification of the transcription terminator; the
full length amino acid sequence of the protein, or a mature form
thereof; the 5' end of the translated region.
[0326] Libraries
[0327] The invention includes nucleic acid or protein libraries
derived from one of the snakes disclosed herein, e.g., a brown,
Taipan inland, Taipan coast, red belly, tiger or rough scale snake.
Nucleic acid libraries can be genomic or cDNA libraries. cDNA
libraries can be derived from particular tissues, e.g., venom gland
tissues. A library will typically include at least 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5 or more diverse members. The nucleic
acid library members can be inserted into vectors, e.g., expression
vectors, e.g., inducible expression vectors.
[0328] Protein library members can be displayed in a number of
ways, e.g., in phage display or cell display systems.
[0329] Arrays and Uses Thereof
[0330] In another aspect, the invention features an array that
includes a substrate having a plurality of addresses. The array can
be a nucleic acid array or a protein array. A nucleic acid array
can display a nucleic acid library from one or more of the snakes
referred to herein. A protein array can display a member of a
protein, polypeptide or peptide library from one or more of the
snakes referred to herein. Proteins or nucleic acids members are
placed at identifiable addressed on the array. The array can have a
density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or
10,000 or more addresses/cm.sup.2, and ranges between. In a
preferred embodiment, the plurality of addresses includes at least
10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a
preferred embodiment, the plurality of addresses includes equal to
or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000
addresses. The substrate can be a two-dimensional substrate such as
a glass slide, a wafer (e.g., silica or plastic), a mass
spectroscopy plate, or a three-dimensional substrate such as a gel
pad. Addresses in addition to address of the plurality can be
disposed on the array.
[0331] In a preferred embodiment, at least one address of the
plurality includes a nucleic acid capture probe that hybridizes
specifically to a member of a nucleic acid library, e.g., the sense
or anti-sense strand. In one preferred embodiment, a subset of
addresses of the plurality of addresses has a nucleic acid capture
probe for a nucleic acid library member. Each address of the subset
can include a capture probe that hybridizes to a different region
of a library member.
[0332] An array can be generated by various methods, e.g., by
photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;
5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow
methods as described in U.S. Pat. No. 5,384,261), pin-based methods
(e.g., as described in U.S. Pat. No. 5,288,514), and bead-based
techniques (e.g., as described in PCT US/93/04145).
[0333] In another preferred embodiment, at least one address of the
plurality includes a polypeptide capture probe that binds
specifically to a SVP polypeptide or fragment thereof. The
polypeptide can be a naturally-occurring interaction partner of SVP
polypeptide. Preferably, the polypeptide is an antibody, e.g., an
antibody described herein (see "Anti-SVP Antibodies," above), such
as a monoclonal antibody or a single-chain antibody.
[0334] Pharmaceutical Compositions
[0335] The invention also provides pharmaceutical compositions that
include a snake venom protease polypeptide of the invention and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. These carriers may be selected from
a non limiting group including sugars, starches, cellulose and its
derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils,
synthetic oils, polyols, alginic acid, phosphate buffered
solutions, emulsifiers, polyethylene glycol and different molecular
weights thereof, isotonic saline and salts such as mineral acid
salts including hydrochlorides, bromides and sulfates, organic
acids such as acetates, propionates and malonates and pyrogen-free
water.
[0336] A useful reference describing pharmaceutically acceptable
carriers, diluents and excipients is Remington's Pharmaceutical
Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated
herein by reference. Supplementary active compounds can also be
incorporated into the compositions.
[0337] The pharmaceutical compositions of the invention can be used
to promote or otherwise facilitate blood coagulation. Examples of
use include administration to bleeding wounds such as during
surgery or following injury or trauma. In one aspect, a snake venom
protease polypeptide is the only blood-coagulating component
present in the pharmaceutical composition. One advantage of
pharmaceutical compositions of the invention is that blood
coagulation occurs rapidly without a need for the sequential or
combinatorial action of plural components such as co-factors. For
example, additional components such as calcium ions, factor Va and
phospholipids are not required. Thus, in some embodiments, the
pharmaceutical composition does not include any co-factors, e.g.,
any of calcium, a phospholipid, factor Va, or vitamin K. In other
embodiments, the pharmaceutical composition can include one or
more, but not all, of calcium, a phospholipid and factor Va.
[0338] In some embodiments, the pharmaceutical composition can
include an additional component or adjuvant. For example, the
composition can include one or more of: an anti-microbial, e.g., an
antibiotic, , an antiviral, an antifungal, an antiparasitic agent,
an anti-inflammatory agent, an antihistamine, an anti-fibrolytic
agent, and a growth factor. Examples of antibiotics include
tetracycline, ciprofloxacin, gentamycin, cyclosporin cefotaxim, and
the like. Examples of antivirals include gangcyclovir, zidovudine,
amantidine, vidarabine, ribaravin, trifluridine, acyclovir,
dideoxyuridine, and the like. Antifungals include, but are not
limited to, diflucan, ketaconizole, nystatin, and the like.
Antiparasitic agents such as pentamidine can be included. The
composition may further include an anti-inflammatory agent such as
.alpha.-1-anti-trypsin, .alpha.-1-antichymotrypsin, and the like.
Examples of growth factors which can be included in the composition
are growth factors that promote the healing of wounds, including,
but not limited to, angiogenins; endothelins; hepatocyte growth
factor and keratinocyte growth factor; fibroblast growth factors,
including fibroblast growth factor-1 (FGF-1), fibroblast growth
factor-2 (FGF-2), and fibroblast growth factor-4 (FGF-4);
platelet-derived growth factors (PDGF); insulin-binding growth
factors (IGF), including insulin-binding growth factor-1 and
insulin-binding growth factor-2; epidermal growth factor (EGF);
transforming growth factors (TGF), including transforming growth
factor-.alpha. and transforming growth factor-.beta.;
cartilage-inducing factors (CIF), including CIP-A and CIP-B;
osteoid-inducing factor (OIF); osteogenin and other bone growth
factors; bone morphogenetic growth factors (BMP), including BMP-1
and BMP-2; collagen growth factor; heparin-binding growth factors,
including heparin-binding growth factor-1 and heparin-binding
growth factor-2; cytokines; interferons; hormones. Other compounds
that can be included in the composition include: vasoconstricting
agents such as adrenalin, or anaesthetics, e.g., local
anaesthetics.
[0339] The pharmaceutical composition can be formulated to promote
stability of the snake venom protease, e.g., to reduce digestion,
e.g., autodigestion, of the snake venom protease. The stability of
the snake venom protease can be promoted, for example, by preparing
providing the snake venom protease in a pharmaceutical composition
having a pH of about 5 to 9, preferably about 6.5 to 7. The
stability of the snake venom protease can also be stabilized by
providing the snake venom protease in a pharmaceutical composition
further includes, e.g., a stabilizer, such as a polyol. In such
embodiments, the pharmaceutical composition can include about 5%,
10%, 20% or more of a polyol (or polyols). An example of a polyol
which can be used in the pharmaceutical composition is glycerol. In
other aspects, the stability of the snake venom protease can be
increased by providing the snake venom protease in a crystallized,
freeze-dried or lyophilized form. If the composition is frozen, the
composition should be thawed prior to the time of use. In another
embodiment, the invention features a composition which includes a
snake venom protease, e.g., a snake venom protease described
herein, and which has a pH of about 5 to 9, preferably about 6.5 to
7. The invention also features a composition which includes a snake
venom protease, e.g., a snake venom protease described herein, and
a stabilizing agent, e.g., a polyol, e.g., glycerol. The polyol can
be present at about 5%, 10% or 20%.
[0340] The dosage of the composition comprising the snake venom
protease depends upon the particular use of the snake venom
protease, but the dosage should be an effective amount for the
composition to perform its intended use. Data obtained from the
cell culture assays and animal studies can be used in formulating a
range of dosage for use in humans. Generally, for a composition
comprising a snake venom protease that is an aqueous solution, it
is believed that from about 1 ml to about 50 ml of such composition
is sufficient to increase fibrin clot formation. However, depending
on the use of the composition, the dosage can range from about 1 ml
to about 200 ml.
[0341] In some embodiments, pharmaceutical compositions of the
invention are topically administered to a wound, surgical incision
or other location where blood loss is to be prevented. To this end,
bandages, patches, gauze, surgical tape, cotton swabs or other
absorbent materials or supportive matrices may be coated,
impregnated or chemically bonded with a composition which includes
a snake venom protease of the invention for topical administration.
Also contemplated are pharmaceutical compositions in the form a
fibrin glue or surgical sealant. Compositions of the invention can
be in the form of creams, lotions, gels, sprays or aerosols for
laparoscopic or open surgical or traumatic wound closure. Topical
administration is desirable in thses applications. In addition,
sutures and staples coated or chemically bonded with a composition
which includes a snake venom protease can be used.
[0342] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0343] It is also contemplated that antifibrinolytic agents may be
added to prevent lysis of the blood clot through the action of
tissue plasminogen activator such as textilinin as described in
International Publication WO 99/58569, aprotinin and EACA.
[0344] Also within the scope of the invention are kits comprising a
snake venom protease or portion thereof described herein. The kit
can include one or more other elements including: instructions for
use; other reagents, e.g., one or more co-factors (e.g., one or
more of calcium, a phospholipid, and factor Va), and/or other
therapeutic agents (e.g., one or more of: an anti-microbial, e.g.,
an antibiotic, an antiviral, an antifungal, an antiparasitic agent,
an anti-inflammatory agent, an antihistamine, an anti-fibrolytic
agent, an analgesic ,and a growth factor); a diluent; devices,
e.g., containers, e.g., sterile containers, or other materials for
preparing the snake venom protease for administration;
pharmaceutically acceptable carriers (e.g., a stabilizer); and
devices or other materials for administration to a subject (e.g.,
syringes, applicators, bandages, spray or aerosol devices). The
instructions can include instructions for therapeutic application
including suggested dosages and/or modes of administration, e.g.,
in a patient with external and/or internal bleeding. In some
applications, the snake venom protease will be reacted with other
components, e.g., one or more co-factor, prior to administration.
In other applications, the snake venom protease can be administered
in combination with other components, e.g., one or more co-factor,
and the kit can include instructions on the amount, dosage, and
timing of administration of the snake venom protease and the other
components.
[0345] In some embodiments, the snake venom protease may be
supplied in lyophilized or freeze dried form. In such embodiments,
the kit can include one or more of: instructions for thawing and/or
hydrolyzing, and a pharmaceutically acceptable carrier or diluent.
In some embodiments, the kit can include instructions for a diluent
or a premeasured amount of a diluent.
[0346] Uses
[0347] The snake venom proteases of the invention have been found
to effectively activate prothrombin by processing prothrombin to
thrombin. Thrombin is a serine protease that cleaves fibrinogen to
generate fibrin, and can act upon several blood factors including
factors V, VIII and XIII to stabilize interaction between fibrin
monomers, thereby enhancing clot formation. Accordingly, the
invention features methods of activating prothrombin and increasing
haemostasis by administering the snake venom proteases described
herein. The method can include: administering a snake venom
protease to a desired site in a subject in an amount effective to
promote or increase fibrin clot formation, to thereby increase
clotting and/or decrease blood or fluid loss. The term "desired
site" refers to a location where the formation of a fibrin clot is
desired. The compositions can be applied directly to the wound,
other tissue or other desired site. Typically for external wounds
it can be applied directly by any means, including spraying the
wound. It can also be applied internally, such as during a surgical
procedure.
[0348] In preferred embodiments, the subject is a mammal, e.g., a
human. Since the snake venom proteases described herein are not
from blood, concerns regarding the risk of blood born pathogens or
other infectious agents which can be found in sealants, adhesives
and hemostats obtained from components of blood are alleviated.
[0349] The snake venom proteases and compositions comprising the
snake venom proteases described herein can be used in various
applications including as a surgical sealant, an adhesive (e.g., a
topical or surgical adhesive), or as a hemstat.
[0350] The methods, kits or pharmaceutical compositions of the
invention can be used, e.g., for connecting tissues or organs,
stopping or reducing bleeding, preventing or inhibiting bleeding,
healing wounds, and/or sealing a wound. The methods, kits and
pharmaceutical compositions can be used in various surgical
settings including: surgery of the nervous system; surgery of the
nose, mouth or pharynx; surgery of the respiratory system; surgery
of the cardiovascular system; surgery of hemic or lymphatic
systems; surgery of the digestive system; surgery of the urinary
system; surgery of the reproductive system; surgery of the
muscloskeletal system; surgery of the integumentary system; plastic
surgery; orthopedic surgery, and transplant surgery. For example,
the snake venom proteases can be used in vascular surgery include
providing hemostasis for stitch hole bleeding of distal coronary
artery anastomoses; left ventricular suture lines; aortotomy and
cannulation sites; diffuse epimyocardial bleeding seen in
reoperations; and oozing from venous bleeding sites, e.g. at
atrial, caval, or right ventricular levels. The subject invention
is also useful for sealing of dacron artery grafts prior to
grafting, sealing tissues outside the body, stopping bleeding from
damaged spleens (thereby saving the organ), livers, and other
parenchymatous organs; sealing tracheal and bronchial anastomoses
and air leaks or lacerations of the lung, sealing bronchial stumps,
bronchial fistulas and esophageal fistulas; and for sutureless
seamless healing ("Zipper" technique). The subject invention is
further useful for providing hemostasis in corneal transplants,
nosebleeds, post tonsillectomies, teeth extractions and other
applications. See G. F. Gestring and R. Lermer, Vascular Surgery,
294-304, September/October 1983. Also, the pharmaceutical
compositions of the invention are especially suited for individuals
with coagulation defects such as hemophilia (e.g., Hemophilia A and
Hemophilia B).
[0351] It has also been found that unlike factor Xa and trocarin,
the snake venom proteases of the invention can activate
descarboxyprothrombin. Descarboxyprothrombin is found, e.g., in
subjects being treated with anticoagulants such as coumadin. Thus,
the methods, kits and pharmaceutical compositions of the invention
can be used to activate prothrombin and increase haemostasis in
subjects being treated with an anticouagulant such as coumadin. The
methods and compositions described herein can be used on these
subjects during surgery or trauma without the need to inhibit or
decrease coumadin treatment.
[0352] As discussed above, the snake venom protease may be
formulated as part of a wound dressing, bandage, patch, gauze,
surgical tape, cotton swabs or other absorbent materials or
supportive matrices. The dressing and bandage are easy-to-use,
requiring no advanced technical knowledge or skill to operate. They
can even be self-administered as an emergency first aid measure.
Such wound dressings and bandages can be used in various field
applications, such as in trauma packs for soldiers, rescue workers,
ambulance/paramedic teams, firemen, and in early trauma and first
aid treatment by emergency room personnel in hospitals and clinics,
particularly in disaster situations. Such dressings may also have
utility in first aid kits for use by the general public or by
medical practitioners. The snake venom protease containing wound
dressing or bandage can further include one or more of calcium, a
phospholipid, a stabilizing agent, or other compound or agent such
as those described herein. For example, the wound dressing or
bandage can further include: an analgesic, an antiviral, an
antifungal, an antiparasitic agent, an anti-inflammatory agent, an
antihistamine, an anti-fibrolytic agent, and a growth factor.
[0353] More than one compound other than the snake venom protease
can be added to the composition, to be released simultaneously, or
each can be released in predetermined time-release manner. The
additional compound (or compounds) added to the composition can be
added at a concentration such that it will be effective for its
intended purpose, e.g., an antibiotic will inhibit the growth of
microbes, an analgesic will relieve pain, etc. In some embodiments,
the dressing or bandage can include an adhesive layer and/or
backing layer. The backing of the dressing or bandage may be of
conventional, non-resorbable materials, e.g., a silicone patch or
plastic material; or it may be of biocompatible, resorbable
materials, e.g., chitin or its derivatives.
[0354] For other applications such as for use as a surgical sealant
or surgical adhesive, the pharmaceutical compositions can in the
form a fibrin glue or surgical sealant that may be in the form of
creams, lotions, gels, sprays, foam or aerosols. For foams, sprays
and aerosols, the composition can be stored in a canister or tank
with a pressurized propellant, so that the components are delivered
to the wound site as an expandable foam or spray. In a preferred
embodiment, the spray, foam or aerosol is provided in a metered
dose. In such embodiments, the methods can include providing a
subject with the spray, aerosol, or foam in a metered dose and
providing the subject with instructions for administering the
spray, aerosol or foam, e.g., to a wound. The instructions can be
for self-administration or administration to others.
[0355] Although the speed with which the composition forms clots
may be to some degree dictated by the application, e.g., rapid
setting for arterial wounds and hemorrhaging tissue damage, slower
setting for treatment of wounds to bony tissue. Preferably,
clotting is evident within ten minutes after application. Most
preferably, clotting will be evident within two to eight minutes
after application.
[0356] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Materials and Methods
[0357] Materials
[0358] A Brown snake venom protease complex was prepared by a
method as described in Masci et al., 1988, Biochem. Int. 17 825,
incorporated herein by reference. 4 mg/ml of prothrombin activator
was stored in 50% glycerol at -20.degree. C. Sephacryl S-300 was
obtained from Amersham Pharmacia Biotech., Uppsala, Sweden, and the
synthetic chromogenic substrate S-2222 was obtained from
Chromogenex, Stockholm, Sweden. Outdated citrated plasma was
obtained from normal, virus-screened volunteers made available by
Princess Alexandra Hospital Blood Bank. Hampton 1 and 2 screen kits
were obtained from Hampton Research, United States of America.
Wizard 1 and 2 screen kits were obtained from Emerald
Biostructures, United Kingdom.
[0359] Brown Snake venom protease Purification
[0360] ConA-Sepharose 4B
[0361] The first step in the purification of P. textilis-snake
venom protease was to isolate Brown snake venom protease complex
from crude venom, as described in Masci et al., 1988, supra. Con A-
Sepharose 4B was packed into a 2.5.times.16 cm column, washed as
recommended by the manufacturer and equilibrated with starting
buffer (0.05 M Tris-HCl, pH 7.4). P. textilis venom (233 mg dry
weight) was reconstituted in 10 ml starting buffer and placed into
a 37.degree. C. water bath until dissolved. The sample was loaded
onto the column and washed with column buffers until the baseline
returned to zero. Elution buffer (0.02 M methyl .alpha.-D
mannopyranoside in 0.05 M Tris-HCl) was applied to the column to
elute bound protein (Brown snake venom protease complex) from the
Con A-Sepharose 4B. The flow rate of the column was 52 ml/hour. The
UV dual wavelength detector was set at 280 mm with attenuations of
0.32 and 0.64 absorbance units full scale (AUFS). Fractions with
S-2222 hydrolytic activity were pooled and concentrated in an
Amicon concentrator, model 405, with a YM3 membrane, having a flow
rate of 48 mL/hour. Purified Brown snake venom protease complexwas
stored in 50% glycerol at -20.degree. C.
[0362] Brown Snake Venom Protease Purification from Brown snake
venom protease Complex
[0363] Sephacryl S-300 chromatography
[0364] Sephacryl S-300 chromatography gel was washed as recommended
by the manufacturer. An 87 cm.times.2.5 cm column of Sephacryl
S-300 was packed at 6.degree. C., and equilibrated with starting
buffer (0.05 M Tris-HCl buffer, pH 7.4), followed by the
equilibration with two column volumes of the same buffer with added
0.8 M NaSCN prior to application of sample. 10 ml of 4 mg/ml
prothrombin activator and 10 ml of 1.6 M NaSCN was incubated for 10
min and loaded onto the column. A Gilson peristaltic pump was set
up with a purple/black chamber, in order to give a flow rate of 40
ml/hr. An Altex UV dual wavelength detector, set at A.sub.280 with
an attenuation of 0.32 AUFS, with a Cole Palmer 2 pen chart
recorder, set at 1 cm/hr were used. Fractions were collected using
time base at time intervals of 10 min/tube initially, followed by a
change to 12 min/tube giving 6.5 and 8 ml fractions respectively,
using a LKB 7000 fraction collector. Chromogenic assays, as
described above, were performed to assess fractions with hydrolytic
activity, which were pooled and concentrated in an Amicon
concentrator, model 42, with a YM3 membrane. This procedure was
repeated three times.
[0365] Superdex 200 Gel Chromatography
[0366] Superdex 200 high resolution gel chromatography was also
used to purify protease from Brown snake venom protease complex.
The Superdex 200 was washed as recommended by the manufacturer,
packed into a 2.5.times.90 cm column, and equilibrated with column
buffer (0.05 M Tris-HCl, pH 7.4, 0.8 M NaSCN). A solution
comprising 9 mL of 5.6 mg/mL Brown snake venom protease complex and
9 mL 1.6 M NaSCN was incubated for 30 min, then loaded onto the
column. The flow rate was 48 mL/hour. The attenuation of the
wavelength detector at 280 mn was 0.32 or 0.64 AUFS. Fractions with
S-2222 activity were pooled and concentrated in an Amicon
concentrator, model 52, with a YM3 membrane. The pooled
concentrated sample (5 mL) was then rechromatographed on the same
column. The final protease preparation was dialyzed overnight in
0.05 M Tris-HCI, pH 7.4, to remove NaSCN from the solution. This
preparation (stored in 10% glycerol/Tris buffer at -20.degree. C.)
was used for all functional and structural characterization
studies.
[0367] High performance liquid chromatography (HPLC)
[0368] Reverse-phase HPLC was performed 25.degree. C., using a
Waters (TM) system consisting of a 6000A dual piston pump and M45A
pump, a 490 wavelength detector set at A.sub.280 nm, and a Wisp
sample injector and a Phemonenex Jupiter C.sub.18-column (KHO-4154)
(1.4 mm.times.250 mm). Chromatography was carried out using a
linear gradient mode over 60 min with a starting solution, (A) 0.1%
TFA in distilled water and eluted with (B) 80% acetonitrile in (A).
Waters Millenium version 1.01 software was used to manage the
system and integrate the data.
[0369] Sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis (PAGE)
[0370] SDS PAGE was performed essentially as described by Laemlli,
1970, Nature 227 680. SDS-PAGE samples were boiled for 10 min in
SDS sample buffer in the presence or absence of
.beta.-mercaptoethanol (.beta.-Me). Gels were stained with
Coomassie blue and destained with methanol, acetic acid and water
(45:10:45).
[0371] N-terminus amino acid sequencing
[0372] Sequencing was performed using the Edman Degradation method.
An Applied Biosytems Procine 492cLC Protein Sequencing System was
used to sequence the Brown snake venom serine protease. Refer to
Applied Biosystems Manual, part no. 904 244, revision D for details
of equipment. Searches were then performed using ExPAsy/NCBI blast
to identify sequence homology between the reptilian serine protease
and Factor Xa, and the T. carinatus Factor Xa-like serine
protease.
[0373] First-Strand cDNA Synthesis and amplification of cDNA
ends
[0374] 1 .mu.g of total RNA isolated from snake gland was used for
cDNA synthesis. For preparation of 5'RACE-Ready cDNA we used 5'-CDS
[5'-(T).sub.25N.sub.-1N-3'; N=A, C, G, or T; N.sub.-1=A, G, or C]
[SEQ ID NO: 32] and SMART II A oligo [
5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3'] [SEQ ID NO: 33] primers from
SMART RACE cDNA Amplification Kit, and for preparation of 3'RACE
ready cDNA -3'-CDS primer A [
5'-AAGCAGTGGTATCAACGCAGAGTAC(T).sub.30N.sub.-1N-3'; N=A,C,G, or T;
N.sub.-1=A,G, or C] [SEQ ID NO: 34] and PowerScript Reverse
Transcriptase from the same Kit. Both cDNA were diluted by adding
100 .mu.l of water and used for Rapid Amplification of cDNA Ends
(RACE) according to the protocol described in User Manual (SMART
RACE cDNA Amplification Kit, Clontech).
[0375] For 3'RACE PCR: 3'RACE cDNA, UPM [Universal Primer Mix A
5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3' (long) [SEQ ID
NO: 35] and 5'-CTAATACGACTCACTATAGGGC-3' (short) [SEQ ID NO: 36]
and degenerate GSP-2 (forward) primer [AAYGGWATGGAYTGYAA;
Y=C+T,W=A+T] [SEQ ID NO: 37] based on the N-terminal amino acid
sequence IVNGMD. Advantage 2 Polymerase Mix (Clontech) was used to
prime the reaction. Thermal Cycler:1 cycle:95.degree. C. 1 min; 25
cycles: 95.degree. C. 30 sec, 65.degree. C. 1 min, 68.degree. C. 3
min; 1 cycle: 68.degree. C. 3 min. Main PCR-product (1.5 kbp) was
isolated from gel using QIAquick Gel extraction Kit ( Qiagen) and
cloned in pGEM-T Easy Vector. After screening the colonies
mini-preps from 35 colonies were isolated using QIAprep Spin
Miniprep Kit (Qiagen).
[0376] DNA sequencing
[0377] DNA sequencing was performed using BigDye Terminator and
forward primer to pGEM-T Easy Vector (GTTTTCCCAGTCACGAC) [SEQ ID
NO: 38]. Only 2 clones not containing stop codon within ca 500 bp
were discovered. These clones were sequenced with For2 primer
(ATCGTTAGTGGATTTGG) [SEQ ID NO: 39]. Stop codon was discovered. The
full sequence of these two clones was similar and the length of
3'-DNA from GSP-2 until first stop codon was 776 bp.
[0378] Using 3'cDNA sequence the reverse primer GSP-1 was designed:
GAAATCGTCTCGGTCTCATTA [SEQ ID NO: 40]. For 5'RACE PCR 5'cDNA, UPM
(see above), GSP-1 and Advantage 2 Polymerase Mix (Clontech) was
used. PCR conditions were the same as for 3'RACE PCR. Main PCR
product (1 kbp) was isolated and cloned in pGEM-T Easy Vector. From
15 clones selected for sequencing 6 were the same, and did not
contain stop codons. Two sequencing primers were used: forward to
pGEM-T Easy Vector (see above) and reverse primer GSP-1. All six
clones contained ATG and were 628 bp from start to the position
corresponding to GSP-2 primer sequence. 3' and 5' cDNA sequences
were used to design forward and reverse primers for full-length
cDNA: SE(forward) ATGGCTCCTCAACTACTCCTCTG [SEQ ID NO: 41] and
SE(reverse) TTAGAGCCGACCAGTGCTTGACTC [SEQ ID NO: 42]. PCR-product
(1.407 bp) was cloned in pGEM-T Easy Vector for sequencing.
[0379] Chromogenic Prothrombin Activation Assays for Brown snake
venom protease complex
[0380] A series of assays were performed to obtain standard curves
for a rate of S-2222 hydrolysis verses an amount of Brown snake
venom protease complex or Brown snake venom protease. Respective
dilutions of Brown snake venom protease complex (4 mg/ml) and
protease (1 mg/ml) varying from {fraction (1/10)} to {fraction
(1/10,000)} were made in 0.05 M Tris-HCl, pH 7.4 and stored on
ice.
[0381] Hydrolytic activity of P. textilis serine protease or the
Brown snake venom protease complex on S-2222 was determined by
equilibration of 0.93 ml of 0.05 M Tris-HCl buffer, pH 7.4, with or
without 10 mM CaCl.sub.2 and 50 .mu.l of 3.0 mM S-2222 in the 1 ml
cell of a Hitachi 557 spectrophotometer at 25.degree. C. The
reaction was initiated by addition of varying concentrations of 20
.mu.l of protease (0.4 mg/ml). The release of p-nitroaniline was
monitored at 405 nm. Assays with 0.91 ml of 0.05 M Tris-HCl buffer,
pH 7.4, containing 0.8 M NaSCN, 50 .mu.l of S-2222 and 40 .mu.l of
0.4 mg/ml Brown snake venom protease complex were performed at time
intervals of 0, 1, 2, 5 and 10 minutes. One unit of activity is
equivalent to the hydrolysis of 1 .mu.mol of substrate/min.
[0382] Prothrombin Activation Assays for Brown Snake Venom
Protease
[0383] Brown snake venom protease (5 .mu.g) was added to 2 mL 0.25
mg/mL prothrombin (in 0.05 M Tris-HCl, pH 7.4). Alquots (20 .mu.L)
of this solution were taken at various time intervals and
chromogenic assays with the thrombin-selective substrate S-2238
were performed. These assays consisted of 930 .mu.L 0.05 M
Tris-HCl, pH 7.4, 50 .mu.L S-2238 and the 20 .mu.L sample. The rate
of substrate hydrolysis was measured at 405 nm. Two 20 .mu.L
aliquots were also taken at each time interval for SDS PAGE
analysis.+-..beta.-mercaptoethanol.
[0384] Clotting Assay
[0385] Citrated plasma clotting assays were performed using a
Hyland-Clotek machine as described by Austen & Rhymes In: A
laboratory manual of blood coagulation. Blackwell Scientific
Publishers, Oxford UK 1975. The assays consisted of 100 .mu.l of
0.05 M Tris-HCl buffer, pH 7.4, 100 .mu.l of citrated human plasma
and 20 .mu.l of a varied concentration of protease. Identical
assays were also performed with or without 0.04 M CaCl.sub.2, and
with 0.8 M NaSCN taking aliquots at time intervals.
[0386] Fibrin Formation in Citrated Plasma by Brown Snake Venom
Protease
[0387] Human citrated plasma (970 .mu.l) was mixed with:
[0388] (1) 20 .mu.l 1.16 mg/mL protease;
[0389] (2) 20 .mu.l 1.16 mg/mL protease and 10 .mu.l 4 M CaCl.sub.2
to give a final Ca.sup.2+ concentration of 40 mM (concentration of
free Ca.sup.2+.about.10 mM);
[0390] (3) 10 .mu.l 4 M CaCl.sub.2.
[0391] Each solution was made up to 1 mL by the addition of 0.05 M
Tris-HCl, pH 7.4. The three solutions were left for 4 hours and the
resulting clots were pressed and washed several times with
dH.sub.20 to remove other plasma proteins from the fibrin clots.
The clots were then added to Eppendorf tubes containing 500 .mu.L
4.times.SDS sample buffer with .beta.-mercaptoethanol and 4 M urea.
An additional drop of .beta.-mercaptoethanol was added to each
Eppendorf tube and left overnight. The samples were boiled for 5
min and 10 .mu.L of each run on a SDS PAGE acrylamide gel as
described herein.
[0392] Active Site Labelling of Brown Snake Venom Protease Complex
and Brown Snake Venom Protease
[0393] Samples (120 .mu.l) of solutions Brown snake venom protease
complex (4 mg/mL) and Brown snake venom protease (2 mg/mL) were
reacted with 15 .mu.L 40 mM DNS-GGACK (4 mM final concentration in
0.05 M Tris-HCl, pH 7.4) for 1 hour. The samples were then dialysed
overnight with a magnetic stirrer at 4.degree. C. in 0.05 M
Tris-HCl, pH 7.4, to remove excess inhibitor. SDS PAGE was then
performed with and without .beta.-mercaptoethanol on both labelled
and unlabelled Brown snake venom protease complex and protease. The
gel with active site labelled proteins was visualized under
ultraviolet light, whereas the other gel was stained with Coomassie
Blue.
[0394] Fibrin Glue studies
[0395] Outdated citrated plasma (3.5 ml) was dispensed into 20 ml
conical plastic vials at 37.degree. C. water bath. 20 .mu.l of 2
mg/ml Brown snake venom serine protease was added to both vials.
0.025 M CaCl.sub.2 was added to one and saline was added to the
other vial. Clotting time was monitored visually and when firm
clots formed they were placed on no. 54 filter paper and pressed.
The resulting pressed clots were extensively washed in distilled
water and stored overnight at 4.degree. C. The clots were
photographed to review texture.
RESULTS
[0396] As shown herein, and exemplified by P. textilis, the snake
venom protease complex comprises a protease characteristic of a
Factor Xa-like scrine protease and a number of other proteins with
unknown function. The isolated snake venom proteases from P.
textilis, O. scutellatus, N. scutatus, T. carinatus and P.
porphyriacus may be useful for the preparation of a pharmaceutical
composition in the form of a topical fibrin "glue" or
"sealant".
[0397] Some of the experiments herein have been performed using P.
textilis derived samples and proteins. However, it will be
appreciated by a person skilled in the art that these experiments
are examples characterising a snake venom protease complex and
snake venom protease that may be applicable to the other snake
venom proteases of the invention.
[0398] Purification of Snake Venom Proteases
[0399] Purification of Brown snake venom protease Complex
(ConA-Sepharose 4B)
[0400] The first step in the purification of P. textilis-snake
venom protease was to isolate Brown snake venom protease complex
from crude venom. A method based on that described by Masci et al,
1988, supra was used. An elution profile at 280 nm resulting from
chromatography of 233 mg dry weight of crude P. textilis venom on
ConA-Sepharose 4B is shown in FIG. 1 (a trace of original
chromatogram).
[0401] The venom was resolved into two major protein peaks, one
that bound to ConA-Separose 4B and had activity against the factor
Xa substrate S-2222 (indicated by line at A in FIG. 1). Based on
A.sub.280 measurements, the activity peak represented approximately
30% of total venom protein.
[0402] Results of SDS PAGE of the pooled Brown snake venom protease
complex concentrate from ConA-Sepharose 4B chromatography are shown
in FIG. 2; lane 1: Molecular weight markers (sizes are shown in
kDa), lane 2: Brown snake venom protease complex without
.beta.-mercaptoethanol, lane 3: Brown snake venom protease complex
with .beta.-mercaptoethanol.
[0403] Arrow A indicates an intact Brown snake venom protease band
in lane 2, whereas arrows B and C designate respective heavy and
light chains of Brown snake venom protease in lane 3 (see
below).
[0404] Brown snake venom protease complex, in the absence of
.beta.-mercaptoethanol (lane 2), comprises a single dominant broad
protein band at .about.150-200 kDa, and three other major bands
with molecular masses of .about.60, 50 and 45 kDa. Summing the
approximate masses of the three major bands in lane 2 results in a
predicted calculated mass of 300-350 kDa for the intact
complex.
[0405] Brown snake venom protease complex in the presence of
.beta.-mercaptoethanol (lane 3) separates into several protein
bands with respective apparent molecular masses of 110, 93, 80, 55,
46, 40 and a broad band (possibly a doublet) at .about.32-34 kDa.
The differences between lanes 2 and 3 indicate that disulfide bonds
appear to link some of the polypeptides in the complex
together.
[0406] The protease component of the Brown snake venom protease
complex exists as a visible doublet in lane 2 at .about.50-60 kDa,
as indicated by arrow A. The heavy chain of the protease presents
as a band at about 40 kDa (indicated by arrow B), and the light
chain of the protease has an approximate mass of 32 kDa (indicated
by arrow C). This designation of the SDS PAGE bands A, B and C was
confirmed by the isolation and characterization experiments
described herein. Some of the bands in FIG. 2 may represent venom
impurities in the Brown snake venom protease complex.
[0407] Purification of the Protease Component from the Brown Snake
Venom Protease Complex
[0408] Sephacryl S-300 Chromatography
[0409] To isolate the Brown snake venom Factor Xa-like serine
protease component of the Brown snake venom protease complex, it
was necessary to dissociate the complex. Speijer et al (1986)
showed 0.8 M NaSCN could efficiently dissociate the O.
scutellatus-prothrombin activator, but never attempted to purify it
with 0.8 M NaSCN in the chromatography procedure. To illustrate an
ability to dissociate the Brown snake venom protease complex with
0.8 M NaSCN, the following experiments were performed and the
results are provided in FIG. 3. 0.8 M NaSCN added to Brown snake
venom protease complex caused a rapid decrease in the citrated
plasma clotting activity from less than 10 sec to greater than 60
sec, however, most S-2222 activity was essentially retained.
[0410] Brown snake venom protease complex treated with 0.8 M NaSCN
was separated into individual components by gel filtration
chromatography on a Sephacryl S-300 column, equilibrated with a 0.8
M NaSCN containing buffer.
[0411] Fractions 30-43 showed S-2222 hydrolysis activity. The
fraction volume was increased for the remaining chromatography
steps from 6.5 ml/tube to 8 ml/tube to reduce the number of
fractions. A second Sephacryl S-300 chromatography was performed
with pooled and concentrated fractions 30-43. S-2222 hydrolytic
activity was observed in fractions 25-29. A third Sephacryl S-300
chromatography with the pooled and concentrated fractions 25-29.
Essentially it gave a single protein peak having S-2222 hydrolytic
activity in fractions 25-29. A high degree of homogeneity was
confirmed by HPLC (FIG. 4). Based on HPLC, the Brown snake venom
serine protease is greater than 95% pure.
[0412] Tables 1-6 summarise purification results and
characterisation of samples from sets of experiments.
[0413] SDS PAGE.+-..beta.-Me of Sephacryl S-300 gel Filtration
Products
[0414] SDS PAGE was performed with pooled fractions from all
chromatography steps, shown in FIG. 5. Lane 4 (containing Sephacryl
S-300, chromatography step 1, pooled fractions 30-43) shows a
homogenous preparation of pooled Brown snake venom serine protease
was not attained since a contaminant exists at a molecular weight
of approximately 107 kDa. Lane 5 (containing Sephacryl S-300,
chromatography step 2, pooled fractions 25-29) shows a greater
percentage of a 55-56 kDa component but still containing a
contaminant requiring a third chromatography. Lanes 6-8, with
varying quantities of the Sephacryl S-300 pooled fractions 25-29
from the third chromatography step, show a homogeneous preparation.
The molecular weight of the intact Brown snake venom serine
protease appears to be between 55 and 56 kDa seen in Lanes 5-8.
[0415] The Brown snake venom serine protease has been compared with
both whole P. textilis venom (Lane 2) and intact Brown snake venom
protease complex, with (Lane 10) and without .beta.-Me (Lane 3).
This showed the position of Brown snake venom serine protease
within the complex and in whole venom.
[0416] Lane 9 of FIG. 5 shows reduction of the Sephacryl S-300
pooled fractions 25-29 from chromatography step 3, with .beta.-Me.
A single band with a molecular weight of approximately 31 kDa can
be seen. A second gel separation was performed to identify the
expected two bands that should have resulted from reduction of the
Brown snake venom serine protease. This gel is shown in FIG. 6.
[0417] SDS PAGE of Sephacryl S-300 pooled and concentrated
fractions 25-29, with or without .beta.-Me, can be seen in FIG. 6.
Lanes 3 (containing Sephacryl S-300, chromatography step 3, pooled
fractions 25-29), 4 and 6 (containing Sephacryl S-300 pooled and
concentrated fractions 25-29 from chromatography 3) shows a
homogeneous preparation of Brown snake venom serine protease was
achieved. However, both Lanes 3 and 6 bands were very faint. The
molecular weight of the Brown snake venom serine protease appears
to be between 55 and 56 kDa, corresponding with the result in FIG.
5.
[0418] Lane 5 (containing Sephacryl S-300 pooled and concentrated
fractions 25-29 from chromatography 3 with .beta.-Me) shows that
the Brown snake venom serine protease contains 3 subunits, however
the last band could be a dye front, which is often seen with the
Laemlli method, or it could be a product of self digestion. Lane 7
(comprising Sephacryl S-300, chromatography step 3, pooled and
concentrated fractions 25-29+.beta.-Me) shows no band and Lane 8
(comprising Sephacryl S-300 pooled and concentrated fractions 25-29
from chromatography 3+.beta.-Me) shows that the Brown snake venom
serine protease is comprised of heavy and light chains. It is
assumed that the Brown snake venom serine proteases comprise heavy
and light chains based on the corresponding Factor Xa and O.
scutellatus serine protease structure. The molecular weight of the
Brown snake venom serine protease heavy chain appears to be
approximately 31 kDa, corresponding with the result in FIG. 5, and
the light chain about 18 kDa. P. textilis whole venom (Lane 2) and
intact Brown snake venom protease complex with .beta.-Me (Lane 9)
was included in the gel so a comparison could be made with the
bands representing Brown snake venom serine protease.
[0419] Superdex 200 Gel Filtration
[0420] In an attempt to improve the purification of Brown snake
venom protease, a higher resolution gel filtration medium (Superdex
200) was alternatively used instead of Sephacryl S-300. The elution
profiles at 280 nm after chromatography and rechromatography of
Brown snake venom protease complex on Superdex 200 in the presence
of NaSCN are shown in FIGS. 10A and 10B. FIGS. 10A and 10B show an
elution profile after chromatography of Brown snake venom protease
complex (18 mL; 50.4 mg) on a column (2.5.times.90 cm) of Superdex
200 in 0.05 M Tris-HCl, pH 7.4 with 0.8 M NaSCN. FIG. 7A shows
chromatography step 1 and FIG. 7B shows chromatography step 2. At
each step fractions with S-2222 activity were pooled and
concentrated, designated by line at A.
[0421] Samples from purification of Brown snake venom protease with
Superdex 200 were separated by SDS PAGE after each purification
step as shown in FIG. 7C. Lanes 1 and 2: pooled concentrate from
chromatography step 1 with (lane 2) and without (lane 1)
.beta.-mercaptoethanol; lanes 3 and 4: pooled concentrate from
chromatography step 2 with (lane 5) and without (lane 4)
.beta.-mercaptoethanol; lane 5: molecular weight markers (sizes are
shown in kDa); arrows A, B and C indicate impurities in lane 4.
[0422] The specific activity of the starting material used in the
Superdex 200 purification was substantially less than that of the
starting material used in the Sephacryl S-300 chromatography (Table
2). This may reflect different activities of different venom
samples. The final product from Superdex 200 purification had a
specific activity of 1.1 U/mL/A.sub.280, less than half the 2.4
U/mL/A.sub.280 of the Sephacryl S-300 product.
[0423] Other methods of isolation are contemplated including
ion-exchange chromatography, urea as an alternative dissociating
agent, purification of the Brown snake venom protease from crude P.
textilis venom using a one step ConA-Sepharose 4B purification
procedure, affinity based on substrate specificity of the protease
and other methods known in the art. The following are examples of
suitable methods for isolating a prothrombin activating protein of
the invention, exemplified with isolation of Brown snake venom
protease. Tables 3-6 show properties of samples during purification
at different steps.
[0424] Protocol 1
[0425] ConA-Sepharose (07-01-03)
[0426] Starting buffer, 0.05 M Tris-HCl, pH 7.4
[0427] Eluting buffer, 0.05 M Tris-HCl, 0.02 M
methyl-.alpha.-D-mannopyran- oside
[0428] Loading sample: dry venom (weight: 541 mg) from Venom
Supplies was reconstituted in 10 ml starting buffer
[0429] A280 of 1 ml solution: 13.5
[0430] Total A280 units loaded: 135
[0431] Activity of sample: 38 U/ml
[0432] Total activity units loaded: 377
[0433] Fractions with S-2222 activity pooled
[0434] 280 of concentrated pool was 6.8 and consisted of 10 ml.
[0435] Total A280 units pooled: 68
[0436] Activity of pool: 2.6 U/ml
[0437] Total activity units pooled: 26.0
[0438] Superdex 200 (13-01-03)
[0439] Starting buffer, 0.05 M Tris-HCl, pH 7.4, 0.8 M NaSCN
[0440] Loading sample: part of the pooled and concentrated peak
from above ConA-Sepharose chromatography with added 0.8 M NaSCN
(A280 8.9, 10 ml, 3.25 U/ml)
[0441] Total A280 units loaded: 89
[0442] Total activity units loaded: 32.5
[0443] Fractions with S-2222 activity pooled
[0444] A280 of concentrated pool was 0.350 and consisted of 20
ml
[0445] Total A280 units pooled: 7
[0446] Activity of concentrated pool: 0.46 U/ml
[0447] Total activity units pooled: 9.2
[0448] Superdex 200 (14-01-03)
[0449] Starting buffer, 0.05 M Tris-HCl, pH 7.4, 0.8 M NaSCN
[0450] Loading sample: pooled and concentrated fractions from
previous Superdex 200 chromatography (A280 0.350, 20 ml, 0.46
U/ml)
[0451] Total A280 units loaded: 7
[0452] Total activity units loaded: 9.2
[0453] Fractions with S-2222 activity pooled
[0454] A280 of concentrated pool was 0.076 and consisted of 40
ml
[0455] Total A280 units pooled: 3.0
[0456] Activity of concentrated pool: 0.11 U/ml
[0457] Total activity units pooled: 4.4
[0458] Protocol 2
[0459] ConA-Sepharose (21-01-03)
[0460] Starting buffer, 0.05 M Tris-HCl, pH 7.4
[0461] Eluting buffer, 0.05 M Tris-HCl, 0.02 M
methyl-.alpha.-D-mannopyran- oside, then 0.05 M Tris-HCl, pH 7.4,
0.8 M NaSCN
[0462] Loading sample: dry venom (weight: 432 mg) from John Weigel
was reconstituted in 10 ml starting buffer
[0463] 280 of Iml solution: 25.6
[0464] Total A280 units loaded: 256
[0465] Activity of sample: 102.9 U/ml
[0466] Total activity units loaded: 1028
[0467] Fractions with S-2222 activity pooled
[0468] 2 pools were made
[0469] 1. concentrated fractions eluted with
methyl-.alpha.-D-mannopyranos- ide (applied to phenyl-sepharose
column)
[0470] A280 of concentrated pool was 0.95 and consisted of 22
ml
[0471] Total A280 units pooled: 20.9
[0472] Activity of pool: 5.0 U/ml
[0473] Total activity units pooled: 110
[0474] 2. concentrated fractions eluted with NaSCN (only half of
this was applied to two identical Superdex 200 chromatography steps
below).
[0475] 200 column as described below
[0476] A280 of concentrated pool was 1.85 and consisted of 27
ml.
[0477] Total A280 units pooled: 68
[0478] Activity of pool: 2.6 U/ml
[0479] Total activity units pooled: 26.0
[0480] Superdex 200 (29-01-03 and 30-01-03)
[0481] Starting buffer, 0.05 M Tris-HCl, pH 7.4, 0.8 M NaSCN
[0482] Loading sample: part of the pooled and concentrated peak
from above ConA-Sepharose chromatography. Two identical
chromatography steps were performed. A loading sample consisted of
16 ml of the pooled and concentrated peak from above ConA-Sepharose
chromatography with added 0.8 M NaSCN:
[0483] A280 of Iml solution: 1.2
[0484] Total A280 units loaded: 19.2
[0485] Activity of sample: 15.7 U/ml
[0486] Total activity units loaded: 250.6
[0487] Fractions with high and identical specific activity from
each of the chromatography steps were pooled and concentrated
(other fractions also had S-2222 activity but the specific activity
was lower, these were pooled separately):
[0488] A280 of concentrated pool was 1.9 and consisted of 9 ml
[0489] Total A280 units pooled: 17.1
[0490] Activity of concentrated pool: 25.7 U/ml
[0491] Total activity units pooled: 231.3
[0492] Superdex 200 (04-02-03)
[0493] Starting buffer, 0.05 M Tris-HCl, pH 7.4 (without NaSCN)
[0494] Loading sample: pooled and concentrated fractions from
previous Superdex 200 chromatography (A280 1.9, 9 ml, 25.7
U/ml)
[0495] Total A280 units loaded: 17.1
[0496] Total activity units loaded: 231.3
[0497] Fractions with S-2222 activity pooled (results below include
fractions with the highest S-2222 activity, other fractions also
had S-2222 activity and these were pooled separately)
[0498] A280 of concentrated pool was 1.7 and consisted of 9.5
ml
[0499] Total A280 units pooled: 16.2
[0500] Activity of concentrated pool: 17.7 U/ml
[0501] Total activity units pooled: 168.2
[0502] Protocol 3
[0503] ConA-Sepharose (10-02-03)
[0504] Starting buffer, 0.05 M Tris-HCl, pH 7.4
[0505] Eluting buffer, 0.025 M Tris-Acetate, pH 6.5, 4 M Urea
[0506] Loading sample: dry venom (weight: 557 mg) reconstituted in
25 ml starting buffer
[0507] A280 of Iml solution: 28
[0508] Total A280 units loaded: 700
[0509] Activity of sample: 83.4 U/ml
[0510] Total activity units loaded: 2087
[0511] Fractions with S-2222 activity pooled
[0512] A280 of pool was 0.592 and consisted of 640 ml.
[0513] Total A280 units pooled: 379
[0514] Activity of pool: 0.152 U/ml
[0515] Total activity units pooled: 97.3
[0516] CM-Sepharose (12-02-03)
[0517] Starting buffer, 0.025 M Tris-Acetate, pH 6.5, 4 M Urea
[0518] Loading sample: pooled fractions from ConA-Sepharose
chromatography (A280 0.592, 640 ml, 0.152 U/ml)
[0519] Total A280 units loaded: 379
[0520] Total activity units loaded: 97
[0521] Once entire sample was loaded onto the column a 0-0.5 M NaCl
gradient was applied
[0522] Fractions with S-2222 activity pooled
[0523] A280 of concentrated pool was 4.5 and consisted of 17.5
ml.
[0524] Total A280 units pooled: 79
[0525] Activity of concentrated pool: 1.44 U/ml
[0526] Total activity units pooled: 25
[0527] Superdex 200 (13-02-03)
[0528] Starting buffer, 0.05 M Tris-Acetate, pH 6.5
[0529] Loading sample: pooled and concentrated fractions from
CM-Sepharose chromatography (A280 4.5, 17.5 ml, 1.44 U/ml)
[0530] Total A280 units loaded: 79
[0531] Total activity units loaded: 25
[0532] Fractions with S-2222 activity pooled (results below refer
to a pooled symmetrical peak, other fractions had S-2222 activity
also)
[0533] A280 of concentrated pool was 0.330 and consisted of 7.5
ml
[0534] Total A280 units pooled: 2.5
[0535] Activity of concentrated pool: 0.146 U/ml
[0536] Total activity units pooled: 1
[0537] Protocol 4
[0538] Phenyl-Sepharose (15-02-03)
[0539] Starting buffer, 0.8 M NaSCN-Phosphate, pH 6.5
[0540] Loading sample: pooled and concentrated fractions from
ConA-Sepharose chromatography (A280 0.95, 22 ml, 5.03 U/ml)
[0541] Total A280 units loaded: 20.9
[0542] Total activity units loaded: 110
[0543] Once entire sample was loaded onto the column a 0.8-0 M
NaSCN gradient was applied
[0544] Fractions with S-2222 activity pooled
[0545] A280 of concentrated pool was 0.485 and consisted of 9.5
ml
[0546] Total A280 units pooled: 4.6
[0547] Activity of concentrated pool: 1.4 U/ml
[0548] Total activity units pooled: 13
[0549] Superdex 200 (18-02-03)
[0550] Starting buffer, 0.05 M Tris-Acetate, pH 6.5
[0551] Loading sample: pooled and concentrated fractions from
phenyl-sepharose chromatography (A280 0.485, 10 ml, 1.4 U/ml)
[0552] Total A280 units loaded: 4.85
[0553] Total activity units loaded: 14
[0554] Fractions with S-2222 activity pooled (two pools were made,
the one described below comprises fractions with greatest
activity)
[0555] A280 of concentrated pool was 0.327 and consisted of 3.5
ml
[0556] Total A280 units pooled: 1.14
[0557] Activity of pool: 1.83 U/ml
[0558] Total activity units pooled: 6.4
[0559] Characterisation of P. textilis-snake venom protease
complex
[0560] Effect of Ca.sup.2+ on Hydrolysis of S-2222 chromogenic
substrate by Brown Snake Venom Protease Complex
[0561] To determine the snake venom protease complex Factor Xa-like
cleavage specificity, chromogenic assays using the Factor Xa
specific chromogenic substrate S-2222 were performed. Brown snake
venom protease complex hydrolyses S-2222, with or without added
Ca.sup.2+. The initial rates of hydrolysis without Ca.sup.2+ are
similar to those in the presence of Ca.sup.2+, but only at
concentrations greater than 2 .mu.g/ml of Brown snake venom
protease complex (data not shown).
[0562] The rate of S-2222 hydrolysis by Brown snake venom protease
complex was approximately linear with an amount of Brown snake
venom proteasecomplex in the assay (as indicated by R.sub.2 values
in Table 7; graphs not shown).
[0563] Added Ca.sup.2+ or Ca.sup.2+ with P.sub.L did not
substantially affect hydrolysis of S-2222 by Brown snake venom
protease complex, which is similar for isolated Brown snake venom
protease. A comparison of S-2222 hydrolysis by Brown snake venom
protease complex with Brown snake venom protease shows that the
rates in Units .mu.g.sup.-1 are similar. Since only about 10-15% of
Brown snake venom protease complex is protease (on a mass basis),
the rate of S-2222 hydrolysis by protease in the Brown snake venom
protease complex in molar terms is about 10 times greater than for
the isolated protease.
[0564] Citrated Plasma Clotting by Brown Snake Venom Protease
Complex
[0565] Citrated plasma clotting assays were performed with Brown
snake venom protease complex to compare clotting properties with
isolated Brown snake venom protease. The results of these
experiments are shown in Table 8. Values shown in Table 8 are
derived from data in relation to clotting of citrated plasma by
Brown snake venom protease complex with and without accessory
components (i.e. Brown snake venom protease complex alone, Brown
snake venom protease complex with 40 mM CaCl.sub.2, and Brown snake
venom protease complex with 40 mM CaCl.sub.2 and phospholipid).
[0566] The results show that Ca.sup.2+ and P.sub.L do not affect
the clotting efficiency of Brown snake venom protease complex.
[0567] Effect of Ca.sup.2+ on citrated plasma clotting time of
Brown snake venom serine protease
[0568] To investigate the clotting properties of Brown snake venom
protease, citrated plasma clotting times without Ca.sup.2+ were
compared to that when Ca.sup.2+ was present. The results in Tables
9 and 10 show that Brown snake venom protease complex does not
require Ca.sup.2+ to clot blood. For example, 39 .mu.g/mL of
isolated Brown snake venom serine protease will clot citrated
plasma in the absence of Ca.sup.2+ in less than 30 sec. Addition of
Ca.sup.2+ resulted in a 200 fold decrease in the amount of Brown
snake venom protease required to give a clotting time of 70 sec
(Table 10). This shows that Brown snake venom protease can convert
prothrombin to thrombin in the absence of Ca.sup.2+ and that
Ca.sup.2+ may facilitate prothrombin cleavage.
[0569] FIGS. 11A-11C show clotting of citrated plasma by Brown
snake venom protease with and without accessory components (data
points are means of duplicate measurements). FIG. 8A: Brown snake
venom protease alone, FIG. 8B: Brown snake venom protease with 10
mM CaCl.sub.2 and FIG. 8C: Brown snake venom protease with 10 mM
CaCl.sub.2 and phospholipid (platelin).
[0570] Ca.sup.2+ would also enhance activation of fibrinogen by
Brown snake venom protease produced thrombin (Mankad and Codispoti,
2001, Am J Surg 182 21S) and accordingly addition of Ca.sup.2+
affecting clotting may be secondary to prothrombin activation.
P.sub.L could also function to facilitate prothrombin cleavage by
Brown snake venom protease, resulting in a further 10 fold decrease
in the amount of Brown snake venom protease required for clotting,
as shown in Table 14.
[0571] Effect of Ca.sup.2+ on cleavage of S-2222 chromogenic
substrate byprothrombin activating proteins
[0572] To determine the Brown snake venom protease complex Factor
Xa-like cleavage specificity, chromogenic assays using the Factor
Xa specific chromogenic substrate S-2222 were performed. S-2222 is
a synthetic chromogenic substrate developed for factor Xa (Aurell
et al., 1977, Thrombin Res 11 595). Hydrolysis of S-2222 releases
p-nitroaniline that is detectable by an increase in absorbance at
405 nm. Plots of enzyme activity versus amount of Brown snake venom
protease were essentially linear, as shown in FIGS. 12A-12D. The
results indicate that the rate of S-2222 hydrolysis was not
affected by the presence of Ca.sup.2+, or Ca.sup.2+ and P.sub.L,
and therefore, that the catalytic site is not affected by Ca.sup.2+
and P.sub.L. From the slope of 0.002 U/.mu.g protease, the specific
activity of the purified preparation was 2 U/mg.
[0573] FIGS. 12A-12D show hydrolysis of S-2222 by Brown snake venom
protease with and without accessory components (data points are
means of duplicate measurements). FIG. 9A: Brown snake venom
protease alone; FIG. 9B: Brown snake venom protease with 10 mM
CaCl.sub.2; FIG. 9C: Brown snake venom protease with 10 mM
CaCl.sub.2 and P.sub.L. and FIG. 9D: slope and R.sub.2 value of
each plot shown in respective FIGS. 12A-12C. R.sub.2 value is the
correlation coefficient for a straight line.
[0574] Brown snake venom protease hydrolyses S-2222, with or
without added Ca.sup.2+ as shown in Table 11 albeit at slightly
lower initial rates of hydrolysis without Ca.sup.2+ compared to
those in the presence of Ca.sup.2+
[0575] In contrast, hydrolysis of a synthetic factor Xa substrate
by Textarin was enhanced by the presence of Ca.sup.2+ and P.sub.L
(Stocker et al., 1994, Toxicon 32 1227), as was that by Trocarin,
the factor Xa-like serine protease from Rough-scaled snake venom
(Joseph et al., 1999, Blood 94 621).
[0576] Isolated Brown Snake Venom Protease Activation of
prothrombin
[0577] Not being bound by theory, it is believed that clotting
occurs by a two-step reaction: (1) conversion of prothrombin to
thrombin by Brown snake venom protease, followed by (2) cleavage of
fibrinogen to fibrin and the activation of factor XIII by
thrombin.
[0578] Referring to FIG. 10 which demonstrates Brown snake venom
serine protease activation of prothromobin, within 10 minutes of
reaction Brown snake venom protease acts to convert prothrombin to
thrombin sufficiently to decrease citrated plasma clotting time
from 65 seconds to a 12 second baseline.
[0579] Prothrombin Activation by Brown snake venom Protease
[0580] The results of the experiments below show that Brown snake
venom protease is able to convert prothrombin to thrombin without
Ca.sup.2+, P.sub.L or accessory proteins like factor Va.
[0581] Results of the S-2222 assays indicate that Brown snake venom
protease may hydrolyse the same bonds as factor Xa in prothrombin.
An effect of Brown snake venom protease on prothrombin was
determined using human prothrombin (0.5 nig in 2 mL 0.05 M Tris-HCl
buffer) reacted with 5 .mu.g Brown snake venom protease (1:100
enzyme: substrate). Reaction products were analysed by non-reducing
SDS PAGE, as shown in FIG. 11A. Additionally, the rate of thrombin
formation was monitored by S-2238 hydrolysis, as shown in FIG. 11B.
S-2238 is commonly used for determining enzyme activity of thrombin
(Komalik and Blomback, 1975, Nature 227 680), incorporated herein
by reference.
[0582] FIG. 11A shows SDS PAGE of the time course of prothrombin
cleavage by Brown snake venom protease. A protein band at .about.40
kDa (lane 5) indicates that thrombin (molecular mass 36.7 kDa) is a
major end product. This protein band increases in intensity with
time showing that prothrombin (PT) is being converted by Brown
snake venom protease to thrombin (T). The prothrombin is
substantially gone by the 48 hour time point (lane 5). FIG. 11B
shows initial activity against S-2238 was very low and increased
approximately 20 fold. From the SDS PAGE gel, it would have been
expected that S-2238 activity would have reached a maximum by 48
hours.
[0583] The human prothrombin used in these experiments was not
totally pure, as indicated by bands shown in lane 2 of FIG. 11A.
Only a prothrombin (PT) band at 72 kDa should be seen (Mann, 1976,
Methods Enzymol 1976 132). A fainter protein band at .about.55 kDa
indicates the presence of some prethrombin 1 (PT.sub.1), possibly
resulting from cleavage of prothrombin by thrombin, as shown in
FIG. 12. Prethrombin 1 is not an active enzyme, confirmed by the
S-2238 assay on the prothrombin solution at t=0.
[0584] A prethrombin 1 band appears to have increased with time
then decreased. Possibly thrombin was present in the prothrombin
solution, but was not detectable by the S-2238 assay. More
probably, thrombin generated during the incubation could have been
responsible for the formation of prethrombin 1.
[0585] To assist with interpreting the results, a mechanism of
prothrombin activation by Brown snake venom protease has been
proposed and a schematic diagram is shown in FIG. 12. The invention
is not bound by this diagram.
[0586] Isolated Brown snake venom A protease activation of
prothrombin and formation of cross-linked fibrin
[0587] From the above results, Brown snake venom protease activates
prothrombin to thrombin. The activated thrombin should sequentially
convert fibrinogen to fibrin. To investigate this, citrated plasma
was incubated with Brown snake venom protease with or without
Ca.sup.2+. This resulted in formation of clots that were washed and
then separated by SDS PAGE, along with a washed fibrin clot formed
by the addition Ca.sup.2+ alone to citrated plasma (representing
formation of a normal in vivo clot since Ca.sup.2+ alone activates
the coagulation cascade. The results of this experiment, shown in
FIG. 13, demonstrates that fibrin produced by the action of Brown
snake venom protease has a similar structure to normal fibrin,
formation of cross-linked fibrin occurs in response to Brown snake
venom serine protease activation of thrombin and resultant Factor
XIII activation.. Approximate clotting times of each experiment
were also recorded (Table 12).
[0588] Using the molecular weight standards (lane 1), and the chain
structures of both fibrinogen (lane 5) and the Ca.sup.2+ produced
fibrin clot (lane 4) from FIG. 13, the bands can be identified. A
band at about 100 kDa in lanes 2 and 3 (Brown snake venom protease
without and with Ca.sup.2+ respectively) is indicative of
.gamma.-dimer (.gamma.-.gamma.). .gamma.-Dimer has a molecular mass
of 105 kDa and results from covalent crosslinks made between two
.gamma.-monomers by factor XIIIa (McKee et al., 1970, Proc Natl
Acad Sci 66 738).
[0589] Bands at approximately 70 and 60 kDa can also be seen in
these lanes indicative of the .alpha.-monomer (.alpha.) and
.beta.-monomer (.beta.) chains of fibrin respectively.
.alpha.-Monomer has a molecular mass of 73 kDa, while .beta.
monomer has a molecular mass of 60 kDa (McKee et al., 1970, supra).
The band with a molecular mass of greater than 400 kDa (top of gel)
is indicative of .alpha.-polymer (.alpha..sub.p), resulting from
lysine-glutamic acid covalent crosslinking of .alpha.-monomer by
factor XIIIa (Gaffney and Brasher, 1974, Nature 251 53). The
.alpha.-chain degradation product (.alpha..sub.1) can also be seen
at .about.38 kDa in lanes 2-4.
[0590] It appears that thrombin resulting from action of Brown
snake venom protease converts fibrinogen to fibrin in a similar
manner as normal .alpha.-thrombin. This is shown by comparing the
banding patterns of the clot produced in the normal way (by
addition of Ca.sup.2+ to citrated plasma) with clots produced by
Brown snake venom protease, with and without Ca.sup.2+ (lanes 3 and
2 respectively). A larger amount of non-crosslinked .alpha.-monomer
is present in the clot produced with Brown snake venom protease
alone (lane 2) compared with in the presence of Ca.sup.2+ (lane 3).
This suggests that factor XIIIa was not as active in formation of
the former clot. This is consistent with the literature since
factor XIIIa activated in the presence of Ca.sup.2+ is more active
than the same enzyme activated in the absence of Ca.sup.2+ (Turner
and Maurer, 2002, Biochemistry 41 7947). Crosslinking of
.alpha.-monomer by factor XIIIa is a slower process than
.gamma.-chain crosslinking, explaining why the .gamma.-chain
appears to be fully crosslinked in all three clots. Leaving the
clot for longer than four hours may have allowed the
.alpha.-monomer to be completely crosslinked.
[0591] Very similar banding patterns were observed in the clot
produced using Brown snake venom protease with Ca.sup.2+ and the
clot representing normal in vivo formation (Ca.sup.2+ alone). There
was a difference however in the clotting times of these two clots
(Table 12). The clot with Brown snake venom protease and Ca.sup.2+
clotted .about.30 times faster than the clot with Ca.sup.2+ alone.
This indicates that clotting was due to the action of Brown snake
venom protease on citrated plasma rather than of the Ca.sup.2+.
Added calcium slightly decreased the clotting time of citrated
plasma by Brown snake venom protease (120 to 60 sec). This is
consistent with the results of citrated plasma clotting assays with
Brown snake venom protease and added Ca.sup.2+.
[0592] Structural Characterization of P. textilis-Snake Venom
Protease Active Site Labelling of Brown snake venom Protease
[0593] Dansyl-L-glutamyl-glycyl-L-arginyl chloromethyl ketone
(DNS-GGACK) is an inhibitor that specifically alkylates the active
site histidine of serine proteases, including factor Xa, thereby
inactivating them (Kettner and Shaw, 1981, Methods Enzymol 80 826).
To determine which SDS PAGE band or bands comprises a catalytic
site, Brown snake venom protease and intact Brown snake venom
protease complex were respectively incubated with DNS-GGACK and
separated run by SDS PAGE. Fluorescent properties of DNS-GGACK
allows visualization of the Brown snake venom protease bands
incorporating covalently bound inhibitor using ultraviolet light.
The results of this experiment are shown in FIG. 14.
[0594] A prominent fluorescent band is visible in lane 3,
corresponding to the intact Brown snake venom protease (lane 7). In
the presence of .beta.-mercaptoethanol (lane 4), the fluorescent
inhibitor was exclusively incorporated into the heavy chain of the
venom protease (band at approximately 37 kDa in lane 8). This shows
that the active site of Brown snake venom protease is located on
the heavy chain rather than the light chain. These results and also
the location of Brown snake venom protease within the Brown snake
venom protease complex banding pattern are confirmed in lanes 1 and
2, and 7 and 8.
[0595] The heavy chain of mammalian factor Xa comprises an enzyme
active site (Bock et al., 1989, Arch Biochem Biophys 273 375).
Analysis of peptide digests of factor Xa inactivated by DNS-GGACK
has shown that histidine 42 of the heavy chain forms part of the
active site. By sequence alignment, the active site histidine
residues of both Trocarin and Brown snake venom protease are
proposed to be in an identical position to the active site
histidine of factor Xa, as shown in FIG. 15. The proposed histidine
of the active site is shown in bold text.
[0596] N-terminal amino acid sequencing of the Brown snake venom
serineprotease, and sequence homology with Factor Xa and T.
carinatus Factor Xa-like serine protease.
[0597] N-Terminal amino acid sequencing of the putative light and
heavy chains of Brown snake venom protease was performed. Short
sequences were also required to facilitate cloning of the cDNA for
Brown snake venom protease from a P. textilis venom gland cDNA
library.
[0598] Brown snake venom protease complex and Brown snake venom
protease were separated by SDS PAGE in the presence of
.beta.-mercaptoethanol and transferred to a PVDF membrane. From
this membrane, sequencing of protein bands was performed.
[0599] Initially, partial amino acid sequence was obtained from the
heavy chain of Brown snake venom protease: IVNGMD(C)KLGE [SEQ ID
NO: 43]. Note that the (C) means that this cycle was blank and
indicates that a cysteine was present but is not certain. The
presence of this cysteine residue was subsequently confirmed after
sequencing of a corresponding cDNA.
[0600] The heavy chain of Brown snake venom protease was a first
protein band transferred to a PVDF membrane and sequenced. The
N-terminus of the heavy chain fragment comprises an amino acid
sequence: IVNGMDCKLGE [SEQ ID NO: 43]. A homology search showed
that this sequence is 100% identical with the N-terminal sequence
of the heavy chain of Trocarin (see FIG. 16). This sequence was
used to design a nucleic acid primer that was used successfully to
amplify Brown snake venom protease cDNA. Similarity was also found
between the N-terminal sequence of Brown snake venom protease and
human factor Xa heavy chain, shown in FIG. 17.
[0601] The light chain of Brown snake venom protease was also amino
acid sequenced. The N-terminal sequence from the band corresponding
to the light chain was ANSLVXXFKSGNI [SEQ ID NO: 44]. The "X"
indicate that there were blanks in the 6.sup.th and 7.sup.th
sequencing cycles. This indicated that the amino acids were either
cysteines, which degrade during sequencing, or that the residues
contained post-translational modifications. The amino acid sequence
of Brown snake venom protease deduced from a nucleotide sequence of
the corresponding cDNA revealed that the "X" amino acid residues
were both glutamic acid. The "X" in the amino acid sequence were
substituted for these residues. Homology of the sequenced
N-terminus of the light chain of the invention was aligned with
Trocarin as shown in FIG. 18. Similarity was also found by aligning
the partial Brown snake venom light chain sequence with the
N-terminal sequence of mouse factor Xa light chain as shown in FIG.
19. The alignments shown in FIGS. 18-23 show that Brown snake venom
protease shares homology with Trocarin, and factor Xa.
[0602] Sequence homology was also found with another second
sequence for Brown snake venom serine protease and Factor Xa.
Homology is greater than 55%.
[0603] A comparison between trocarin amino acid sequence and
N-terminal sequence obtained from Brown snake venom serine
protease.
[0604] The full length cDNA and encoded protein sequence of Brown
snake venom serine protease was obtained as described above and
both sequences are shown in FIGS. 25-30.
[0605] A comparison of the complete amino acid sequence of Brown
snake venom serine protease and trocarin is shown in FIGS. 26 and
27. The overall level of sequence identity was 81%, however there
are a number of unique features in Brown snake venom protease
beginning at the N-terminal propeptide sequence (40 amino acids)
which is not present in trocarin. It was predicted that the
propeptide cleavage site to be between R and A at the end of the
propeptide as shown in FIG. 29. This is supported by a BLAST search
which reveals a series of haemostatic factors including factors X,
IX, VII and others and their precursors as being related to Brown
snake venom serine protease. This sequence at the end of the
propeptide KRANS - - - EE - - - EREC and additional glutamic acid
residues important for function in binding Ca.sup.2+ are well
conserved. Indeed there are several blocks of sequence conserved
including the cleavage site or parts of it between the heavy and
light chains RIVNGMD [SEQ ID NO:45] just distal to amino acid
residue 200.
[0606] Another difference with trocarin evident in the alignment is
the presence of 28 amino acids in Brown snake venom protease
(residues 182-209) which are absent in trocarin. This sequence
leads up the predicted cleavage site between light and heavy chains
as shown in FIGS. 27A and 29. The light chain of Trocarin consists
of 141 residues and ends with the amino acid sequence KARNK [SEQ ID
NO: 46] (Joseph et al., 1999, Blood 94 621). The predicted amino
acid sequence of Brown snake venom protease light chain comprises a
similar sequence (KTRNK) [SEQ ID NO: 47] starting at amino acid 176
of FIG. 29. The light chain of Brown snake venom protease may be
cleaved at this point thereby removing the final 28 amino acids
before the start of the heavy chain. The molecular mass of Brown
snake venom protease was calculated to be 43,587 Da, assuming
cleavage at the above indicated point, and respective heavy and
light chains are predicted to have a molecular mass of 27,952 and
15,652 Da (see Table 13).
[0607] Distance migrated of proteins separated by SDS PAGE was also
used to estimate the molecular mass of Brown snake venom protease
and its component chains (data not shown). Approximate molecular
masses of the intact Brown snake venom protease and its heavy and
lights chains were determined to be 53 kDa, 35 kDa and 29 kDa
respectively based on SDS PAGE data (see Table 13).
[0608] The cDNA nucleotide sequence does not indicate whether a
protein is cleaved or if it has post-translational modifications.
For this reason, Trocarin was used as a model since the amino acid
sequence of native Trocarin (determined by protein sequencing) and
the translated cDNA nucleotide sequence of Brown snake venom
protease are very similar. The molecular mass of native Trocarin
was estimated to be 46,515 Da (Joseph et al., 1999, supra). The
molecular mass calculated from the Trocarin amino acid sequence
without any post-translational modifications is about 42,455 Da.
Accordingly, there is approximately 4,060 Da of post-translational
modifications including Glu residues, N-glycosylation and
O-glycosylation. Trocarin and Brown snake venom protease are very
similar and therefore it may be predicted that Brown snake venom
protease will have a similar post-translational modification as
trocarin. Based on this assumption, the molecular mass of Brown
snake venom protease with post-translational modifications and a
cleaved light chain is 47,647 Da, which is consistent with the
experimentally determined value of 53 and 48 kDa. Factor Xa has a
molecular mass of 46 kDa (Di Scipio et al., 1977, Biochemistry 67
99). This calculated mass of 47,647 Da was used in determining the
concentration of Brown snake venom protease in solution.
[0609] Comparison of snake Derived Venom Protease Proteins
[0610] The venom glands from a coastal taipan, inland taipan,
brown, tiger, red-belly black and rough scale snake were removed
from alive road damaged specimens, and total RNA extracted via the
TRI Reagent.COPYRGT. method for RNA extraction (Sigma, Castle Hill,
Australia). First-strand cDNA was then synthesised from the RNA.
The cDNA was then screened for the factor Xa-like snake venom
protease gene via PCR using degenerate primers designed from the
preliminary amino acid sequence deduced from the brown snake
protease. Note that different regions of the protease were
amplified, using different primer sets, with focus upon the heavy
chain of the factor Xa-like component. All PCR products were run on
a 1.5% TAE agarose gel, extracted using a QIAEX II gel extraction
kit (Qiagen, Hilden, Germany), cloned into the pGEM-T vector system
(Promega, Annandale, Australia) and subsequently sequenced using an
ABI Prism Big Dye Terminator Cycle Sequence Ready Reaction Kit
(Perkin-Elmer, Boston, U.S.A.). Sequence alignments were then
performed between the proteases isolated from the all five species.
FIG. 27 shows an amino acid alignment of the brown, coastal taipan,
red belly black, tiger and rough scale snake proteases of the
invention with trocarin. FIG. 28 shows an amino acid alignment of
these proteases of the invention with human factor Xa. FIG. 29
shows an alignment of all of the brown, coastal taipan, inland
taipan, red belly black, tiger and rough scale snake proteases of
the invention with propeptide, light chain and heavy chain domains
indicated.
[0611] Cloning and Sequencing of nucleic acids encoding Taipan,
Tiger, Rough Scale and Red-belly Black Snake venom protease
proteins
[0612] Respective full length nucleic acids encoding snake venom
protease proteins were cloned and sequenced from taipan, tiger,
rough scale and red-belly black snakes. An alignment of the
nucleotide sequences of the above snake derived nucleic acids with
the snake venom protease from the common brown snake revealed a
number of points of interest. This includes almost 100% homology
within a 40 amino acid propeptide amino acid sequence (residues
1-40 shown in FIGS. 29 and 30), not withstanding a single amino
acid change within the red-belly black snake. This high degree of
conservation is also observed within the regions of the cleavage
site between the propeptide and the light chain, and the light
chain and the heavy chain (see FIG. 29). Overall there is a 72%
degree of homology between the five snakes. The protease from the
taipan is most closely related to that of the common brown snake,
being 92% homologous, as would be expected as both are group C
prothrombin activators. Likewise, there is a high degree of
similarity between the group D prothrombin activators from the
mainland tiger and rough scale snakes with 95% homology, with the
red-belly black snake protease being the most distinct of the five.
One final point of interest is the area of low homology within the
heavy chain, where deletions are observed within the tiger,
red-belly black and rough scale snakes, plus the premature
termination of the protease eleven amino acids from the end in the
tiger and rough scale snakes.
[0613] There are conserved novel regions of the snake venom
proteases that are distinct from both trocarin and human factor Xa
and all other known proteins. These regions include the following,
which are also shown in FIGS. 27-29 as consensus sequences.
[0614] KREASLPDFVQS (residues 181-192) SEQ ID NO: 19];
[0615] LKKSDNPSPDIR (residues 198-209) [SEQ ID NO: 20]; and
[0616] SVX.sub.1VGEIX.sub.2X.sub.3SR (residues 260-270) [SEQ ID NO:
21]
[0617] X.sub.1, X.sub.2 and X.sub.3 may be any amino acid, but
preferably X.sub.1 is either V or I, X.sub.2 is either D or N and
X.sub.3 is either R or I.
[0618] MAPQLLLCLILTFLWSLPEAESNVFLKSK (residues 1-29) [SEQ ID NO:22]
and
[0619] ANRFLQRTKR (residues 31-40) [SEQ ID NO: 23]
[0620] KREASLPDFVQSXXAXXLKKSDNPSPDIR (residues 181-209) [SEQ ID NO:
24], wherein X may be any amino acid
[0621] MAPQLLLCLILTFLWSLPEAESNVFLKSKXANRFLQRTKR (residues 1-40)
[SEQ ID NO: 25], wherein X may be any amino acid
[0622] It will be appreciated that SEQ ID NOS: 23, 24 and 25
correspond to a predicted propeptide comprising amino acids 1-40 as
shown in FIG. 29 and accordingly may not in one embodiment form
part of a proteolytically digested mature protein.
[0623] A person skilled in the art will be able to identify other
novel conserved regions of the prothrombin activating proteins of
the invention based on alignment data provided in FIGS. 27-29.
[0624] Similarly, novel conserved nucleic acids encoding the
prothrombin activating proteins of the invention may be determined
from alignment data provide in FIG. 30. Such novel nucleic acids
may be useful, for example, in designing specific nucleic acid
primers and/or probes to amplify, sequence and/or identify a
nucleic acid of the invention.
[0625] Fibrin glue
[0626] Citrated plasma with added Brown snake venom scrine protease
clotted very quickly in the both the presence and absence of 10 mM
Ca.sup.2+. The macroscopic texture of the two clots appears to
differ for the two preparations.
[0627] Mouse Tail-Vein Bleeding Model
[0628] Effectiveness of purified Brown snake venom protease
functioning as an anti-bleeding agent was tested in mice using a
tail-vein bleeding model. The results of these experiments are
shown in Tables 14 and 15 and FIGS. 32-33.
[0629] Mouse tail-vein bleeding studies were performed as
essentially described by Masci et al (2000) with minor alteration.
The results are shown in FIGS. 32 and 33 and Tables 14 and 15. P.
textilis protease (250 .mu.L; 65 .mu.g/mL P. textilis protease in
0.02 M Tris-HCl, pH 7.4, 10 mM CaCl.sub.2) was applied topically to
the open wound of the severed tail for 3 minutes. Blood loss was
measured using preweighed eppendorf tubes. Accuracy dictated that
blood loss was measured by weight rather than volume. It is noted
that all mice topically treated with the protease showed a large
clot at the site of injury as shown in FIG. 27. Mice were
cuthanized via cervical dislocation.
[0630] Data for Table 15 and FIG. 28 were obtained from experiments
wherein an open wound of a severed mouse tail was submersed in 250
.mu.l 0.9% sodium chloride (saline control) with or without 65
.mu.g Brown snake venom protease for three minutes. Blood lose was
measured by weight. As Table 15 and FIG. 28 show, cofactors are not
required to clot blood.
[0631] As shown in Tables 14 and 15 and FIG. 28, Brown snake venom
protease significantly reduced blood loss in mice (0.169
g.+-.0.086) compared to the control animals (0.542.+-.0.160) (Mann
Whitney U test, p=0.021) when corrected for technical errors.
EXAMPLE
Generation of a cDNA Library From the Venom Gland of P. textilis to
Establish a Microarray Chip for Cross-Species Comparisons and Use
For Drug Discovery
[0632] Messenger RNA extracted from the venom gland of the target
snake was amplified as cDNA and fragments greater then 600bp in
size cloned into a .lambda.Trip1Ex2 vector using a SMART cDNA
library synthesis kit (Clontech, Palo Alto, U.S.A.). Such a cDNA
library was produced from both the taipan and brown snake, and
preliminary sequence analysis performed on approximately 30
transcripts from each library. This process involved PCR
amplification to detect the presence and size of the insert,
followed by conversion of the .lambda.Trip1Ex2 to a pTrip1Ex2
plasmid and subsequent sequencing.
[0633] Due to its average increased insert size and variation, it
was decided to select the taipan cDNA library for the establishment
of a microarray chip. Subsequently, 4800 cDNA clones were randomly
isolated for large scale PCR amplification and purification, which
were then spotted in duplicate onto coated glass slides using an
GMS 417 array spotter available within the Queensland Institute of
Medical Research. RNA from the venom glands of the afore mentioned
snakes was then amplified in a linear fashion using a modified
Eberwine antisense RNA amplification protocol (yielding up to a
seventy fold increase in RNA concentration) awaiting hybridisation
to the chip.
DISCUSSION
[0634] The snake venom proteases of the invention have a unique
structure and functional properties. They also share some
similarities with Factor Xa and the O. scutellatus-prothrombin
activator. The snake venom proteases of the invention clot citrated
plasma without the presence of Ca.sup.2+. In vivo, Factor Xa also
requires the presence of Ca.sup.2+ for normal clotting.
Accordingly, it is a novel and surprising observation that the
snake venom proteases of the invention are capable of clotting
blood without the presence of factors such as phospholipid, factor
Va or Ca.sup.2+.
[0635] The Factor Xa specific chromogenic substrate, S-2222 is
cleaved by the snake venom proteases of the invention. This shows
that the snake venom proteases have very similar cleavage
specificity to Factor Xa. Furthermore, it is interesting that
Ca.sup.2+ only enhances the rate of S-2222 hydrolysis at
concentrations lower than 2 .mu.g/ml of Brown snake venom protease
complex. Also, when NaSCN is added to the Brown snake venom
protease complex, not all of the S-2222 activity is maintained.
These observations are distinct from the work by Speijer et al
(1986) in relation to the O. scutellatus-prothrombin activator.
[0636] The simple gel filtration method using Sephacryl S-300
proved relatively poor for the isolation of the serine protease
component from the Brown snake venom protease complex, evident from
the number of chromatographies required for purification. Despite
the extended purification, a homogenous preparation was finally
achieved, determined by HPLC and SDS PAGE in the absence of
.beta.-Me.
[0637] The SDS PAGE results suggest that the Brown snake venom
protease has a native molecular weight of between 55 and 56 kDa.
The Brown snake venom protease shares greater size similarity with
the 54 kDa mammalian Factor Xa (Mann et al, 1987) than the 60 kDa
O. scutellatus Factor Xa-like protease (Speijer et al, 1987, J.
Biol. Chem. 261 13258). Furthermore, the Brown snake venom protease
chain structure shows greater resemblance to Factor Xa than the O.
scutellatus Factor Xa-like serine protease.
[0638] SDS PAGE (+.beta.-Me) showed that the Brown snake venom
protease comprises two peptide chains, probably linked together by
a disulfide bridge. This is further supported by the finding of two
N-terminus amino acids from sequencing of the Brown snake venom
protease. From the results, the sizes of the heavy and light chains
are approximately 31 and 18 kDa respectively, however this does not
correspond with a total protease molecular weight of 55-56 kDa. In
contrast to the Brown snake venom protease of the invention, the O.
scutellatus Factor Xa-like serine protease was found to consist of
two chains composed of 30 kDa each (Speijer et al, 1986,
supra).
[0639] It was an interesting observation that 100% sequence
homology exists between the first 11 amino acids of the T.
carinatus Factor Xa-like serine protease and the Brown snake venom
proteases of the invention. This shows that a degree of amino acid
sequence (revealed with the complete amino acid sequence of the
Brown snake venom protease) conservation has occurred throughout
the evolution of these two Australian snake venom prothrombin
activators. Sequence homology also exists between Factor Xa and the
Brown snake venom protease, showing that some amino acids have been
conserved in the evolution of snakes and mammals. However, as also
shown in FIGS. 27 and 28, the snake venom proteases of the
invention have novel conserved regions that are distinct from
Factor Xa and Trocarin and all other proteins known to the
applicant.
[0640] Factor Xa has all the typical characteristics of a serine
protease, having two similarly structured domains, intradomain
disulfide bonds and others (Stubbs & Bode, 1994, supra).
However, serine proteases differences confer their specific
function. For example, the Factor Xa active site cleft is much more
open than the thrombin cleft (Stubbs & Bode, 1994, supra),
which may contribute to the Factor Xa cleavage specificity for
Arg274-Thr275 and Arg323-Ile324.
[0641] A novel therapeutic use for the snake venom proteases of the
invention is as reagents for making topical fibrin glue. The snake
venom proteases of the invention may provide a more effective
therapeutic for preparing fibrin glue than current methods. Topical
fibrin glue prepared with the snake venom proteases of the
invention may greatly reduce haemorrhage experienced in trauma and
hence could possibly save many human and non-human animal lives.
For example, emergency medical units may be equipped with bandages
and the like impregnated with a fibrin glue comprising snake venom
proteases of the invention to prevent bleeding at an accident.
[0642] ABBREVIATIONS
[0643] A405--absorbance at 405 nm
[0644] Arg--arginine
[0645] AUFS--absorbance units full scale at 280 nm
[0646] C--cysteine
[0647] Ca.sup.2+--calcium ions
[0648] CaCl.sub.2--calcium chloride
[0649] cm--centimeter
[0650] D--aspartic acid
[0651] E--glutamic acid
[0652] F--phenylalanine
[0653] G--glycine
[0654] HPLC--high performance liquid chromatography
[0655] hr--hour
[0656] I--isoleucine
[0657] Ile--isoleucine
[0658] K--lysine
[0659] kDa--kilo Dalton
[0660] L--leucine
[0661] M--methionine
[0662] M--molar
[0663] mg--milligram
[0664] min--minute
[0665] ml--milli liter
[0666] mM--mill molar
[0667] N--asparagines
[0668] NaSCN--sodium thiocyanate
[0669] nm--nano meter
[0670] O. scutellatus--Oxyuranus scutellatus
[0671] P. textilis--Pseudonaja textilis
[0672] PAGE--polyacrylamide gel electrophoresis
[0673] PEG--polyethylene glycol
[0674] Q--glutamine
[0675] S--serine
[0676] SDS--sodium dodecyl sulfate
[0677] sec--second
[0678] T--threonine
[0679] T. carinatus--Tropidechis carinatus
[0680] TFA--trifluoroacetic acid
[0681] Thr--threonine
[0682] TOF--time of flight
[0683] V--valine
[0684] Y--tyrosine
[0685] .beta.-Me--.beta.-mercaptoethanol
[0686] .mu.l--micro liter
[0687] .mu.mol--micro molar
5TABLE 1 Sample Total volume Total Activity Specific Activity Yield
Purifi- Step (mL) A280 (Units) (Units/mL/A280) (%) cation Brown
20.0 80.0 106.5 1.3 100 - SVP Complex with NaSCN Step 1 15.0 25.7
51.2 2.0 48.1 1.5 Step 2 8.0 13.0 27.3 2.1 25.6 1.6 Step 3 5.5 7.3
17.2 2.4 16.1 1.8
[0688]
6TABLE 2 SAMPLE TOTAL SPECIFIC VOLUME TOTAL ACTIVITY ACTIVITY YIELD
PURIFI- STEP (mL) A.sub.280 (Units) (Units/mL/A.sub.280) (%) CATION
Brown 18.0 50.4 40.1 0.8 100 -- SVP Complex with NaSCN Step 1 5.0
13.0 13.1 1.0 32.6 1.2 Step 2 3.5 10.4 10.9 1.1 27.2 1.3
[0689]
7TABLE 3 SAMPLE TOTAL SPECIFIC VOLUME TOTAL ACTIVITY ACTIVITY YIELD
PURIFI- STEP (mL) A.sub.280 (Units) (Units/mL/A.sub.280) (%) CATION
Brown 10.0 89.0 32.5 0.4 100 -- SVP Complex + NaSCN Superdex 20.0
7.0 9.2 1.3 28.3 3.25 200 (step 1) Superdex 40 3.0 4.4 1.5 13.5
3.75 200 (step 2)
[0690]
8TABLE 4 SAMPLE TOTAL SPECIFIC VOLUME TOTAL ACTIVITY ACTIVITY YIELD
PURIFI- STEP (mL) A.sub.280 (Units) (Units/mL/A.sub.280) (%) CATION
Brown 32.0 38.4 501.2 13.1 100 -- SVP Complex Superdex 9.0 17.1
231.3 13.5 46.1 1.0 200 (step 1) Superdex 9.5 16.2 168.2 10.4 33.6
0.8 200 (step 2)
[0691]
9TABLE 5 SAMPLE TOTAL SPECIFIC VOLUME TOTAL ACTIVITY ACTIVITY YIELD
PURIFI- STEP (mL) A.sub.280 (Units) (Units/mL/A.sub.280) (%) CATION
Venom 25.0 700 2087 2.97 100 -- ConA 4B 640.0 379.0 97.3 0.257 4.6
0.09 CM- 17.5 79.0 25.0 0.32 1.2 0.12 Sepharose Superdex 7.5 2.5 1
0.442 0.05 0.15 200
[0692]
10TABLE 6 SAMPLE TOTAL SPECIFIC VOLUME TOTAL ACTIVITY ACTIVITY
YIELD PURIFI- STEP (mL) A.sub.280 (Units) (Units/mL/A.sub.280) (%)
CATION Brown 22.0 20.9 110 5.3 100 -- SVP Complex Phenyl- 10 4.85
14 2.9 12.7 0.55 Sepharose Superdex 3.5 1.14 6.4 5.6 5.8 1.1
200
[0693]
11 TABLE 7 CONDITION SLOPE R.sub.2 A-Brown SVP complex alone 0.0022
0.9965 B Brown SVP complex w/ 10 mM CaCl.sub.2 0.0025 0.9884 C
Brown SVP complex w/ 10 mM CaCl.sub.2+ 0.0041 0.9852
phospholipid.
[0694]
12 TABLE 8 B SVP COMPLEX (.mu.g) .+-. CLOTTING TIME ACCESSORY
COMPONENTS (sec) Alone Ca.sup.2+ Ca.sup.2+and P.sub.L 20 0.5 0.5
0.6 10 2.5 3 3
[0695]
13TABLE 9 Brown SVP Clotting time (sec) Clotting time (sec)
(.mu.g/mL -Ca.sup.2+ +Ca.sup.2+ 39.000 27.3 14.9 26.000 35.1 18.7
13.000 38.4 23.4 6.500 51.6 24.0 2.600 >100 27.6 1.300 >100
34.5 0.650 >100 34.7
[0696]
14 TABLE 10 BROWN SVP (.mu.g) .+-. CLOTTING TIME ACCESSORY
COMPONENTS (sec) Alone Ca.sup.2+ Ca.sup.2+and P.sub.L 70 4 0.02
0.002 50 11 0.05 0.004
[0697]
15TABLE 11 Brown SVP .DELTA.A.sub.405/min .DELTA.A.sub.405/min
(.mu.g/mL) -Ca.sup.2+ +Ca.sup.2+ 39 0.70 1.11 19.5 0.33 0.90 13
0.26 0.36 6.5 0.15 0.24 2.6 0.12 0.11 1.3 0.06 0.09 0.65 0.03 0.05
0.33 0.01 0.01
[0698]
16 TABLE 12 CLOT TYPE TIME (SEC) Brown SVP 120 Brown SVP with 40 mM
CaCl.sub.2 60 CaCl.sub.2 alone 1800
[0699]
17 TABLE 12 MOLECULAR MASS (DA) Heavy Light Intact METHOD OF MASS
DETERMINATION chain chain protein SDS PAGE 35000 29000 53000 Mass
spectrometry -- -- 48000 Calculated from cDNA sequence 27952 18789
46723 without propeptide (residues 1-40) Calculated from cDNA
sequence 27952 15652 43587 without propeptide and assuming light
chain has 141 residues as does that of Trocarin Calculated from
cDNA sequence -- -- 47647 without propeptide, 141 residue light
chain, Gla residues and glycosylation at the same level as
Trocarin
[0700]
18TABLE 14 Blood loss (grams) Relative blood conserved Treatment (n
= 2) (%) Saline 0.4335 -- Protease/10 mM Ca.sup.2+ 0.0166 96.17
[0701]
19TABLE 15 BLOOD LOSS BLOOD LOSS TEST (g) CONTROL (g) 1 0.12 1 0.64
2 0.16 2 0.71 3 0.29 3 0.42 4 0.10 5 0.39 Average blood 0.169 g
.+-. 0.086 Average blood 0.542 .+-. 0.160 loss (g) .+-. SD loss (g)
.+-. SD
[0702]
20TABLE 16 Venom concentration Clotting times Snake venoms (mg/mL)
(sec .+-. 0.5 secs) A Pseudonaja textilis- Qld 2.0 3.9 Pseudonaja
textilis- SA 2.0 5.4 Pseudonaja textilis- Goyder lagoon 2.0 8.4
Pseudonaja nuchalis 2.0 8.7 Pseudonaja affinis 2.0 5.5 Pseudonaja
inframacula 2.0 7.9 Oxyuranus scutellatus 200.0 24.1 Oxyuranus
microlepidotus 500.0 19.7 Notechis scutatus 500.0 34.9 Notechis
ater niger 500.0 27.7 Notechis ater serventyi 1,000.0 31.1
Hoplocephalus stephansii 1,000.0 36.2 Pseudechis porphiracus 500.0
48.6 Australaps surperba 1,000.0 38.7 Tropedechis carinatus 500.0
34.9 B Australaps ramsayii 1,000.0 250>clot<600 Pseudechis
guttatus 1,000.0 250>clot<600 Pseudechis australis 1,000.0
>100; no clot Pseudechis colletti 1,000.0 >100; no clot
Acanthopis antarcticus 1000.0 >100; no clot Cryptophis
nigrescens 1,000.0 >100; no clot C Bothrops jararaca 100.0 11.7
Agkistradom rhodasroma 100.0 6.3 Vipera russelli 500.0 >200 Naja
naja 500.0 >200 Naja naja miolepis 500 >200 Echis carinatus
200.0 10.4 Bothrops atrox 100.0 5.3 Bungarus fasciatus 50.0 12.6
Ophiophagus hannah 100.0 >200; weak clot
[0703] Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
It will therefore be appreciated by those of skill in the art that,
in light of the instant disclosure, various modifications and
changes can be made in the particular embodiments exemplified
without departing from the scope of the present invention.
* * * * *
References